Compositions and methods for inducing phagocytosis of MHC class I positive cells and countering anti-CD47/SIRPA resistance

ABSTRACT

Methods and compositions are provided for inducing phagocytosis of a target cell, treating an individual having cancer, treating an individual having an intracellular pathogen infection (e.g., a chronic infection), and/or reducing the number of inflicted cells (e.g., cancer cells, cells infected with an intracellular pathogen, etc.) in an individual. Methods and compositions are also provided for predicting whether an individual is resistant (or susceptible) to treatment with an anti-CD47/SIRPA agent. In some cases, the subject methods and compositions include an anti-MHC Class I/LILRB1 agent. In some cases, the subject methods and compositions include an anti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPA agent (e.g., co-administration of an anti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPA agent). Kits are also provided for practicing the methods of the disclosure.

CROSS-REFERENCE

This application claims benefit and is a Continuation of ApplicationSer. No. 16/394,411, filed Apr. 25, 2019, which is a Continuation ofapplication Ser. No. 15/518,976, filed Apr. 13, 2017, U.S. Pat. No.10,316,094, granted Jun. 11, 2019, which is a 371 application and claimsthe benefit of PCT Application No. PCT/US2015/057233, filed Oct. 23,2015, which claims benefit of U.S. Provisional Patent Application No.62/068,351 filed Oct. 24, 2014, which applications are incorporatedherein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under contracts CA086017and CA139490 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

INTRODUCTION

Programmed cell death (PCD) and phagocytic cell removal are common waysthat an organism responds in order to remove damaged, precancerous, orinfected cells. Cells that survive this organismal response (e.g.,cancerous cells, chronically infected cells, etc.) have devised ways toevade PCD and phagocytic cell removal. For example, growing tumors, andcells harboring an infection, are under constant pressure from the hostimmune system, and evasion of immunosurveillance is critical for theprogression of cancer and chronic infection in patients. Therapeuticagents that disrupt this escape, either by directly stimulating theimmune system to attack tumor cells and/or infected cells, or byblocking immunosuppressive signals expressed by tumor cells and/orinfected cells, comprise a promising new category of drugs.

If properly engaged, effector cells of both the innate and adaptiveimmune systems possess the ability to attack cancer cells and/orinfected cells. For example, tumor-binding monoclonal antibodies caninduce this attack, and efficacy is in part dependent on the antibody'sability to stimulate antibody-dependent cellular phagocytosis (ADCP) bymacrophages. However, CD47, a “don't eat me” signal, is constitutivelyupregulated on a wide variety of diseased cells, cancer cells, andinfected cells, allowing these cells to evade phagocytosis. Althoughbinding of an anti-tumor antibody to tumor cells is sufficient to engagemacrophage Fc receptors and thereby stimulate some degree of tumor cellphagocytosis, the potency of this response is strongly limited by thetumor's expression of CD47.

Anti-CD47/SIRPA agents that block the interaction between CD47 on onecell (e.g., a cancer cell, an infected cell, etc.) and SIRPA on anothercell (e.g., a phagocytic cell) counteract the increase of CD47expression and facilitate the phagocytosis of the cancer cell and/or theinfected cell. For example, CD47 blocking antibodies simultaneouslydisrupt the CD47/SIRPA interaction and opsonize tumor cells to whichthey bind, powerfully promoting macrophage phagocytosis (FIG. 1A).Anti-CD47/SIRPA agents can be used to treat and/or protect against awide variety of conditions/disorders.

However, some cancer cells and/or infected cells are resistant totreatment with anti-CD47/SIRPA agents. The present disclosure providescompositions and methods for predicting whether a cancer (e.g.,predicting whether an individual having cancer) will be responsive toanti-CD47 treatment. This disclosure further provides compositions andmethods for treating a cancer and/or an infection that is resistant totreatment with anti-CD47/SIRPA agents. For example, this disclosureprovides compositions and methods for reducing the resistance totreatment (with an anti-CD47/SIRPA agent) of a cancer cell and/or aninfected cell.

Publications

-   Borges et al., 1997, Journal of Immunology 159, 5192-5196; Fanger et    al., 1998, European Journal of Immunology 28, 3423-3434; Willcox et    al., 2003, Nature immunology 4, 913-919; Cheng, H. et al., 2011, The    Journal of biological chemistry 286, 18013-18025,    doi:10.1074/jbc.M111.221028.

SUMMARY

Methods and compositions are provided for inducing phagocytosis of atarget cell, treating an individual having cancer, treating anindividual having an intracellular pathogen infection (e.g., a chronicinfection), and/or reducing the number of inflicted cells (e.g., cancercells, cells infected with an intracellular pathogen, etc.) in anindividual. Methods and compositions are also provided for predictingwhether an individual is resistant (or susceptible) to treatment with ananti-CD47/SIRPA agent. In some cases, the subject methods andcompositions include an anti-MHC Class I/LILRB1 agent. In some cases,the subject methods and compositions include an anti-MHC Class I/LILRB1agent and an anti-CD47/SIRPA agent (e.g., co-administration of ananti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPA agent). Kits arealso provided for practicing the methods of the disclosure.

In some embodiments, a subject composition (e.g., for increasingphagocytosis of a target cell) includes: (a) an anti-MHC ClassI/LILRB1agent (e.g., an MHC Class I binding agent such as an anti-MHC Class Iantibody or an LILRB1 peptide; an LILRB1 binding agent such as ananti-LILRB1 antibody or a soluble MHC class I complex that binds toLILRB1; and the like); and (b) at least one of: (i) an agent thatopsonizes the target cell, and (ii) an anti-CD47/SIRPA agent. In somecases, the anti-MHC ClassI/LILRB1 agent specifically binds majorhistocompatibility complex (MHC) Class I. In some cases, the anti-MHCClassI/LILRB1 agent is an antibody that specifically binds classical MHCClass I, where the classical MHC Class I lacks HLA-G and comprises atleast one of HLA-A, HLA-B, and HLA-C. In some cases, the anti-MHCClassI/LILRB1 agent is a soluble leukocyte immunoglobulin-like receptorsubfamily B member 1 (LILRB1) peptide. In some cases, the anti-MHCClassI/LILRB1 agent specifically binds leukocyte immunoglobulin-likereceptor subfamily B member 1 (LILRB1) and does not activate signalingthrough LILRB1 upon binding. In some cases, the anti-MHC ClassI/LILRB1agent is an anti-LILRB1 antibody. In some cases, the composition alsoincludes an anti-CD47/SIRPA agent. In some cases, the agent thatopsonizes the target cell is an antibody other than an anti-CD47antibody. In some cases, the composition includes an anti-CD47/SIRPAagent and an agent that opsonizes the target cell.

In some embodiments, a subject kit (e.g., for increasing phagocytosis ofa target cell) includes: (a) an anti-MHC ClassI/LILRB1 agent (e.g., anMHC Class I binding agent such as an anti-MHC Class I antibody or anLILRB1 peptide; an LILRB1 binding agent such as an anti-LILRB1 antibodyor a soluble MHC class I complex that binds to LILRB1; and the like);and (b) an anti-CD47/SIRPA agent and/or an agent that opsonizes thetarget cell (e.g., where (a) and (b) are in separate containers). Insome cases, at least one of (a) and (b) is present as a therapeuticformulation.

In some embodiments, a subject method is a method of inducingphagocytosis of a target cell, and the method includes: contacting atarget cell with a macrophage in the presence of an anti-MHCClassI/LILRB1 agent (e.g., an MHC Class I binding agent such as ananti-MHC Class I antibody or an LILRB1 peptide; an LILRB1 binding agentsuch as an anti-LILRB1 antibody or a soluble MHC class I complex thatbinds to LILRB1; and the like) and at least one of: an anti-CD47/SIRPAagent and an agent that opsonizes the target cell, for a period of timesufficient to induce phagocytosis of the target cell by the macrophage.In some cases, the target cell is a cancer cell. In some cases, thetarget cell is a cell infected with an intracellular pathogen. In somecases, the target cell is a cancer cell of an individual having cancer.In some cases, the contacting is in vitro or ex vivo. In some cases, thecontacting is in vivo. In some cases, the anti-MHC ClassI/LILRB1 agentspecifically binds major histocompatibility complex (MHC) Class I. Insome cases, the anti-MHC ClassI/LILRB1 agent specifically bindsclassical MHC Class I, wherein said classical MHC Class I lacks HLA-Gand comprises at least one of HLA-A, HLA-B, and HLA-C. In some cases,the anti-MHC ClassI/LILRB1 agent is an antibody or a binding fragmentthereof. In some cases, the anti-MHC ClassI/LILRB1 agent is a solubleleukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1)polypeptide. In some cases, the anti-MHC ClassI/LILRB1 agentspecifically binds leukocyte immunoglobulin-like receptor subfamily Bmember 1 (LILRB1) and does not activate signaling through LILRB1 uponbinding. In some cases, the anti-MHC ClassI/LILRB1 agent is ananti-LILRB1 antibody or a binding fragment thereof. In some cases, thecontacting is in the presence of an anti-MHC ClassI/LILRB1 agent and ananti-CD47/SIRPA agent.

In some embodiments, a subject method is a method of treating anindividual having cancer (and/or having an intracellular pathogeninfection) where the method includes administering to the individual:(a) an anti-MHC ClassI/LILRB1 agent (e.g., an MHC Class I binding agentsuch as an anti-MHC Class I antibody or an LILRB1 peptide; an LILRB1binding agent such as an anti-LILRB1 antibody or a soluble MHC class Icomplex that binds to LILRB1; and the like); and (b) at least one of:(i) an anti-CD47/SIRPA agent, and (ii) an agent that opsonizes a targetcell of the individual, where the target cell is a cancer cell (and/or acell harboring an intracellular pathogen), in amounts effective forreducing the number of cancer cells (and/or cells harboring theintracellular pathogen) in the individual. In some cases, (a) and (b)are administered simultaneously. In some cases, (a) and (b) are notadministered simultaneously. In some cases, the method includes, priorto the administering step: measuring the expression level of MajorHistocompatibility Complex (MHC) Class I in a biological sample of theindividual, where the biological sample includes a cancer cell (and/or acell harboring an intracellular pathogen); and providing a prediction,based on the result of the measuring step, that the individual isresistant to treatment with an anti-CD47/SIRPA agent.

In some embodiments, a subject method is a method of predicting whetheran individual is resistant or susceptible to treatment with ananti-CD47/SIRPA agent, where the method includes: (a) measuring theexpression level of Major Histocompatibility Complex (MHC) Class I in abiological sample of the individual, where the biological sampleincludes a cancer cell (and/or a cell harboring an intracellularpathogen), to produce a measured test value; (b) comparing the measuredtest value to a control value; and (c) providing a prediction, based onthe comparing step, as to whether the individual is resistant orsusceptible to treatment with an anti-CD47/SIRPA agent. In some cases,the measuring step includes an antibody-based method. In some cases, theantibody-based method includes flow cytometry. In some cases, the MHCClass I is classical MHC Class I that lacks HLA-G and comprises HLA-A,HLA-B, and/or HLAC. In some cases, the control value is the expressionlevel of MHC Class I from a cell or population of cells known to exhibita phenotype of resistance to treatment with an anti-CD47/SIRPA agent. Insome cases, the control value is the background value of the measuringstep. In some cases, the method includes a step of determining that thecancer cell (and/or the cell harboring an intracellular pathogen) ispositive for MHC Class I, and providing a prediction that the individualis resistant treatment with an anti-CD47/SIRPA agent. In some cases, themethod includes: (a) providing a prediction that the individual isresistant to treatment with an anti-CD47/SIRPA agent, and (b)administering to the individual an anti-MHC ClassI/LILRB1 agent and ananti-CD47/SIRPA agent (e.g., an MHC Class I binding agent such as ananti-MHC Class I antibody or an LILRB1 peptide; an LILRB1 binding agentsuch as an anti-LILRB1 antibody or a soluble MHC class I complex thatbinds to LILRB1; and the like). In some cases, the providing aprediction step includes generating a report that includes at least oneof: (i) the measured expression level of MHC Class I, (ii) thenormalized measured expression level of MHC Class I, (iii) a predictionof resistance or susceptibility to an anti-CD47/SIRPA agent, and (iv) arecommended therapy based on the measured test value. In some cases, thereport is displayed to an output device at a location remote to thecomputer. In some cases, a subject method includes anidentifying/selecting a patient need of co-administration of an anti-MHCClassI/LILRB1 agent and an anti-CD47/SIRPA agent.

Aspects of the disclosure include anti-MHC ClassI/LILRB1 agents. In somecases a subject anti-MHC ClassI/LILRB1 agent is an anti-LLRB1 antibody(e.g., humanized, e.g., IgG₄ isotype humanized antibody). In some casesa subject anti-MHC ClassI/LILRB1 agent is an anti-MHC ClassI antibody(e.g., humanized, e.g., IgG₄ isotype humanized antibody). In some casesa subject anti-MHC ClassI/LILRB1 agent is a polypeptide the includes thelight chain CDR amino acid sequences set forth in SEQ ID NOs: 8-10 andthe heavy chain CDR amino acid sequences set forth in SEQ ID NOs: 12-14.In some cases, the agent includes the light chain amino acid sequenceset forth in SEQ ID NO: 7 and the heavy chain amino acid sequence setforth in SEQ ID NO: 11. In some cases, the agent is a humanized antibodyor a Fab fragment. In some cases, the agent is present in apharmaceutical composition. Aspects of the disclosure also includespreparation of a medicament (e.g., a medicament that includes a subjectanti-MHC ClassI/LILRB1 agent, a medicament that includes a subjectanti-MHC ClassI/LILRB1 agent and an anti-CD47/SIRPA agent, a medicamentthat includes a subject anti-MHC ClassI/LILRB1 agent and an antibodyagainst a cancer antigen, a medicament that includes a subject anti-MHCClassI/LILRB1 agent and an anti-CD47/SIRPA agent and an antibody againsta cancer antigen, and the like). In some cases, an anti-MHCClassI/LILRB1 agent (e.g., in any of the methods or compositions of thedisclosure) is an antibody (e.g., anti-LLRB1 antibody, anti-MHC Class Iantibody) and can be a humanized antibody, e.g., can be an IgG₄ isotypehumanized antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1C. Resistance to macrophage phagocytosis correlates with MHCClass I expression. (FIG. 1A) Cartoon schematic of signaling betweenmacrophages and target cancer cells under untreated conditions (left)and treatment with anti-CD47 therapy (right). (FIG. 1B) FACS-basedmeasurement of phagocytosis by donor-derived human macrophages against apanel of 18 cancer cell lines, including Colon Carcinoma (CC), SmallCell Lung Cancer (SCLC), Breast Carcinoma (BC), PancreaticNeuroendocrine Tumor (PNET), Melanoma (Mel), and Osteosarcoma (OS) upontreatment with PBS or a humanized anti-CD47 antibody, Hu5F9-G4. Allvalues were normalized to DLD1 as an index control. Error bars representthe standard deviation of assays with four independent donors for alllines, with the exception of H128, H1688, and SkBr3, for which theyrepresent three independent donors. 12 of 18 lines show a significantincrease (p<0.05; Student's two-sided t-test without multiplecomparisons correction) in phagocytosis upon treatment with Hu5F9-G4.(FIG. 1C) Log-transformed scatterplot of normalized phagocyticefficiency upon treatment with the anti-CD47 antibody Hu5F9-G4 (Y axis)plotted against surface expression of HLA-A, B, C as measured by FACSanalysis with the pan-HLA binding antibody W6/32 (X axis). Y values arelog-transformed averages of values represented in FIG. 1A. There is asignificant, inverse relationship between HLA-A, B, C expression andsensitivity to phagocytosis upon treatment with Hu5F9-G4 (R²=0.411,p=0.002).

FIG. 2A-2G. MHC class I directly protects cells from macrophage attack.(FIG. 2A) Expression summary table (left) and FACS scatterplot (right)of CD47 and HLA-A, B, C expression levels (Y and X axes, respectively)for four genetically engineered sub-lines of KWNO1. (FIG. 2B) Expressionsummary table (left) and FACS scatterplot (right) of CD47 and HLA-A, B,C expression levels (Y and X axes, respectively) for four geneticallyengineered sub-lines of DLD1. (FIG. 2C) FACS-based measurement ofphagocytosis by human macrophages co-cultured with parental KWNO1 (red)and a B2M-deleted sub-line, KWNO1-ΔB2M (black), upon treatment with PBSor the anti-CD47 antibody Hu5F9-G4. Values are normalized to the maximumlevel of phagocytosis in each independent replicate experiment. Errorbars represent the standard deviation of experiments with eightindependent macrophage donors. ** p<0.01, *** p<0.001, 2-way ANOVA withmultiple comparisons correction. (FIG. 2D) FACS-based measurement ofphagocytosis by human macrophages co-cultured with parental DLD1 (black)and an MHC-reconstituted transgenic sub-line, DLD1-Tg(B2M) (red), upontreatment with PBS or the anti-CD47 antibody Hu5F9-G4. Values arenormalized to the maximum level of phagocytosis in each independentreplicate experiment. Error bars represent the standard deviation ofexperiments with eight independent macrophage donors. n.s., notsignificant. *** p<0.001, 2-way ANOVA with multiple comparisonscorrection. (FIG. 2E) FACS-based measurement of phagocytosis by humanmacrophages co-cultured with KWNO1 genetic variants. Values arenormalized to the maximum level of phagocytosis in each independentreplicate experiment. Anti-EpCam antibody is clone 1B7. Error barsrepresent the standard deviation of experiments from eight independentmacrophage donors. n.s., not significant. * p<0.05, ** p<0.01, ***p<0.001, 2-way ANOVA with multiple comparisons correction. (FIG. 2F)FACS-based measurement of phagocytosis by human macrophages co-culturedwith DLD1 genetic variants. Values are normalized to the maximum levelof phagocytosis in each independent replicate experiment. Anti-EGFR isthe clinical antibody cetuximab. Error bars represent the standarddeviation of experiments from eight independent macrophage donors. n.s.,not significant. * p<0.05, ** p<0.01, *** p<0.001, 2-way ANOVA withmultiple comparisons correction. (FIG. 2G) FACS-based measurement ofphagocytosis by donor-derived macrophages of the MHC− line DLD1 (leftpanel, black), and the MHC+ lines U205, SAOS2, SKMel3, NCI-H196, HCT116,and KWNO1 (right panel, gray) upon treatment with PBS; a fragment ofantigen binding (Fab) generated via proteolytic cleavage of the pan-HLAantibody W6/32; the anti-CD47 antibody Hu5F9-G4; or a combination of theW6/32 Fab and Hu5F9-G4. Values are normalized to the maximum level ofphagocytosis in each independent replicate experiment. Error barsrepresent the standard deviation of experiments with four independentmacrophage donors. n.s., not significant. * p<0.05, Student's t-testwithout multiple comparisons correction.

FIG. 3A-3C. The receptor LILRB1 mediates macrophage detection of MHCclass I. (FIG. 3A) Representative histogram plots from FACS analysis ofLILRB1 and LILRB2 expression in primary human CD14+ peripheral bloodmonocytes (left), and day 7 ex vivo cultured macrophages derived fromthe same donor (right). IgG control is indicated in red. Specificstaining is indicated in blue. (FIG. 3B) LILRB1 and LILRB2 expressionlevels, as measured by mean fluorescence intensity (MFI, left panel) orpercent positive cells (right panel), in 4 pairs of primary monocytes(blue circle) and ex vivo cultured macrophages from the same donors (redtriangle). n.s., not significant. *** p<0.001, 2-way ANOVA with multiplecomparisons correction. (FIG. 3C) FACS-based measurement of phagocytosisby donor-derived macrophages of parental KWNO1 (red) and theMHC-negative sub-line KWNO1-ΔB2M (black) upon treatment with PBS, theanti-CD47 antibody Hu5F9-G4, a fragment of antigen binding (Fab)generated by proteolytic cleavage of the pan-HLA antibody W6/32, theanti-LILRB1 blocking antibody GHI/75, or the anti-LILRB2 blockingantibody 27D6. Values are normalized to the highest level ofphagocytosis observed in a given experimental replicate. Error barsrepresent the standard deviation of assays performed with eightindependent macrophage donors. n.s., not significant. *** p<0.001, 2-wayANOVA with multiple comparisons correction.

FIG. 4A-4C. B2M confers species-specific protection against macrophagephagocytosis. (FIG. 4A) The crystal structure of the LILRB1:B2M:HLA-A2complex, illustrating differences between human and mouse sequences.LILRB1 (magenta) makes extensive contact with residues of B2M (blue) butonly limited contact with HLA-A2 (gray). Inset: residues that differbetween human and mouse B2M, and that are located within 5 angstroms ofLILRB1 are highlighted in orange. These residues were mutated, asindicated, to form a human-mouse chimeric B2m (hmcB2M). Images weregenerated using MacPyMol from published structure data IP7Q (Fanger etal, European Journal of Immunology 28, 3423-3434 (1998)). (FIG. 4B)FACS-based measurement of surface MHC class I expression, as measured bystaining with the pan-HLA-A, B, C binding antibody W6/32. Parental DLD1cells (left panel, black) are negative for surface MHC class I, buttransgenic reconstitution with human B2M (DLD1-Tg(B2M), red), or ahuman-mouse-chimeric B2M (DLD1-Tg(hmcB2M), purple), both induceefficient surface MHC class I expression. Parental KWNO1 (right panel,red) are positive for MHC class I expression, which can be eliminated byCRISPR-mediated deletion of B2M (KWNO1-ΔB2M, black); transgenicexpression of hmcB2M restores surface expression of MHC(KWNO1-Tg(hmcB2M)-ΔB2M, purple). (FIG. 4C) FACS-based measurement ofphagocytosis by primary human donor-derived macrophages (Y axis) or NSGmouse-derived macrophages (X axis) co-cultured with parental DLD1(black); B2M-deleted KWNO1 (KWNO1-ΔB2M, black); a DLD1 sub-line withtransgenic expression of fully human B2M (red); parental KWNO1 (red); aDLD1 sub-line with transgenic expression of a human-mouse chimeric B2M(purple); or a KWNO1 sub-line with deleted B2M and transgenic expressionof hmcB2M (KWNO1-Tg(hmcB2M)-ΔB2M, purple). Vertical error bars representthe standard deviation of experiments with eight independent biologicaldonors, while horizontal error bars represent the standard deviation ofeight experimental replicates across two experiments with independentlyderived NSG (NOD-SCID II2rγ^(−/−)) mouse macrophages.

FIG. 5A-5E. MHC class I expression protects tumor cells from macrophagesin vivo. (FIG. 5A) Schematic of in vivo human macrophage xenograftsystem. Tumor cells and human macrophages were mixed on ice in a 1:2ratio, and antibodies were added as indicated before subcutaneousinjection into the flank of NSG mice. Tumor bioluminescence images fromNSG (NOD-SCID II2rγ^(−/−)) mice post-. (FIG. 5B) Tumor bioluminescenceimages from NSG (NOD-SCID II2rγ^(−/−)) mice post-engraftment withprimary ex vivo differentiated human macrophages and MHC+ tumor cells(KWNO1, left panel) or MHC− tumor cells (KWNO1-ΔB2M, right panel),treated with PBS, the anti-CD47 antibody Hu5F9-G4, the recombinantanti-LILRB1 antibody GHI75-G₄, or a combination of the two antibodies.Five animals were evaluated per treatment group. Bioluminescence imagesare from day 7 (left panel) or day 14 (right panel). (FIG. 5C)Fluorescence microscopy of sections taken from mixed MHC+ and MHC− KWNO1tumors injected subcutaneously into the flanks of NSG mice. ChimericMHC+ cells are marked by GFP expression, while MHC− cells are marked byRFP expression. Mouse macrophages are visualized by F4/80 staining. Ahigh degree of macrophage infiltration into the tumor is evident, andmacrophages can be observed in the act of phagocytosis (whitearrowhead). Scale bar represents 200 μM. (FIG. 5D) Schematic of in vivoexperiment to assess sensitivity of MHC− and chimeric MHC+ cells toanti-CD47 agents. Equivalent numbers of cells were injected into theflanks of NSG mice, and allowed 14 days to engraft. Starting 14 dayspost-injection, the mice received once-weekly injections of either PBSor anti-CD47 antibody. (FIG. 5E) Tumor bioluminescence (total flux,photons/second) of MHC− (KWNO1-ΔB2M) and chimeric MHC+(KWNO1-Tg(hmcB2M)-ΔB2M) cells engrafted subcutaneously in the flanks ofNSG mice, and starting at day 14 post-engraftment, treated once per weekwith either PBS or 10 mg/kg of the anti-CD47 antibody Hu5F9-G4. Linesrepresent the fold change in bioluminescent flux, normalized to theinitial value as measured at day 7. Error bars represent standard errorof the mean of 15 mice per group. Gray is MHC− (KWNO1-ΔB2M) treated withPBS; pink is chimeric MHC+ (KWNO1-Tg(hmcB2M)-ΔB2M) treated with PBS;black is MHC− (KWNO1-ΔB2M) treated with Hu5F9-G4; purple is chimericMHC+ (KWNO1-Tg(hmcB2M)-ΔB2M) treated with Hu5F9-G4. * indicates p<0.05versus vehicle treatment groups, *** indicates p<1e-4 versus vehicletreatment groups, 2-way ANOVA with Tukey's multiple comparisonscorrection; see Supplementary Table 1 for comprehensive statisticalcomparisons. Luminescence measurements were discontinued when mice hadto be euthanized due to tumor growth.

FIG. 6. Gating strategy of FACS-based in vitro phagocytosis assay. Ourin vitro phagocytosis assay relies on differential labeling of targetcancer cells (GFP+ CD45−) and human macrophages (GFP− CD45+) in order toidentify macrophages that have successfully phagocytosed labeled targetcells. Co-incubation with labeled target cells under PBS treatmentconditions (top panels) leads to only minimal emergence of a GFP+ CD45+population, which is measured as a percentage of the total macrophages.In contrast, treatment with a tumor-opsonizing antibody, especiallyunder conditions of CD47 blockade, results in a clear emergence of adistinct GFP+ CD45+ population. We previously validated this assay bypost-assay sorting and microscopy of the populations, which confirmedthe gating strategy as successfully identifying macrophages that hadphagocytosed one or more cancer cells (Weiskopf et al, Science 341,88-91 (2013)).

FIG. 7. CD47 expression does not correlate with sensitivity toHu5F9-G4-induced phagocytosis. Log-transformed scatterplot of normalizedphagocytic efficiency upon treatment with the anti-CD47 antibodyHu5F9-G4 (Y axis) plotted against surface expression of CD47, asmeasured by FACS analysis with the anti-human mouse monoclonal antibodyB6H12 (X axis). There is no significant relationship between these twoparameters, R²=0.094, p=0.217.

FIG. 8. MHC protein expression is high in phagocytosis-resistant celllines. Histogram plots of HLA-A, B, C (top) and B2M expression (bottom)for two colon cancer cell lines: DLD1 (red) and HCT116 (magenta); twosmall cell lung cancer lines, NCI-H82 (blue), and NCI-H196 (purple); anda pancreatic neuroendocrine tumor KWNO1 (green), as measured by theBioLegend LegendScreen FACS-based antibody array system. Isotype controlstain is indicated in blue, while specific stain is indicated in red.

FIG. 9. HLA-A, B, C expression of phagocytosis-resistant cell lines.Log-scale scatterplot of phagocytic efficiency in a panel of cell linesupon treatment with the anti-CD47 antibody Hu5F9-G4 (Y axis), plottedagainst surface expression of HLA-A, B, C as measured by FACS analysiswith a pan-HLA-A, B, C binding antibody (X axis). The data is a subsetof that shown in FIG. 1B, in order to highlight the cell lines used forthe experiment shown in FIG. 2G.

FIG. 10. LILRB1 antibody sensitizes MHC+ DLD1 cells to phagocytosis.FACS-based measurement of phagocytosis by donor-derived macrophages ofparental DLD1 (black) and the human MHC-reconstituted transgenicsub-line DLD1-Tg(B2M) (red) upon treatment with PBS; the anti-CD47antibody Hu5F9-G4; a fragment of antigen binding (Fab) generated byproteolytic cleavage of the pan-HLA antibody W6/32; the anti-LILRB1antibody GHI/75; or the anti-LILRB2 antibody 27D6. Values are normalizedto the highest level of phagocytosis observed in a given experimentalreplicate. Error bars represent the standard deviation of assaysperformed with eight independent macrophage donors. While DLD1-Tg(B2M)is significantly more resistant to Hu5F9-G4-induced phagocytosis thanparental DLD1 (p<0.01, 2-way ANOVA with multiple comparisonscorrection), this significant difference is completely erased upondisruption of the MHC/LILRB1 signaling axis by either W6/32 fab orGHI/75. n.s., not significant. ** p<0.01, *** p<0.001, 2-way ANOVA withmultiple comparisons correction.

FIG. 11. Mouse macrophages express the LILRB family homolog PirB. FACShistogram of ex vivo-differentiated NSG macrophages stained withPE-labeled IgG control (red) or anti-PirA/B antibody 6C1.

FIG. 12. Mouse B2m does not efficiently form stable MHC complexes withhuman HLA alpha chains. FACS histogram of parental DLD1 cells (black),DLD1 cells reconstituted with a human B2M transgene (red), or with amouse B2m transgene (blue). While fully human B2M expression facilitatesrobust surface MHC expression, as detected by the pan-HLA antibodyW6/32, expression of mouse B2m facilitates only low levels of surfaceMHC.

FIG. 13. Human macrophages continue to phagocytose cancer cells daysafter engraftment into NSG mice. KWNO1 cells were mixed with humanmacrophages and either PBS, the anti-CD47 antibody Hu5F9-G4, therecombinant anti-LILRB1 antibody GHI75-G₄, or a combination of the twoantibodies. These mixtures were then co-engrafted subcutaneously intoNSG mice, and tumor luminescence was assessed at 12 hours, 4 days, and 7days post-engraftment. By 12 hours post-engraftment, tumors treated witha combination of Hu5F9-G4 and GHI75-G₄ exhibited a rapid decrease inluminescence as compared to other groups. The luminescence of thesetumors continued to decline between 12 hours and 4 dayspost-engraftment, suggesting a continuation of macrophage phagocytosisof cancer cells. Error bars represent the standard deviation of fivemice per group.

FIG. 14. MHC expression does not impact the in vitro growth kinetics ofthe DLD1 cell line. Growth curve of parental DLD1 (pink) and a DLD1sub-line expressing the human-mouse chimeric B2M (DLD1-Tg(hmcB2M),blue), as measured by cell counting at 1, 2, and 4 days. Error barsrepresent the standard deviation of three independent replicates. Thereare no significant differences between these lines at any time-point.

FIG. 15. MHC expression does not influence the in vitro growth kineticsof the KWNO1 cell line in a mixed co-culture. FACS analysis of MHCexpression in a 50-50 in vitro co-culture of chimeric MHC+ andMHC− KWNO1cells, as measured by pan-HLA binding antibody. After 21 days, there wasno significant change in the proportion of MHC+ andMHC− cells from day0. Error bars represent the standard deviation of three independentco-cultures

FIG. 16A-16B. A Table that provides a statistical comparison betweentime points and groups.

FIG. 17. Results from experiments that measure phagocytosis of twodifferent human breast cancer cell lines (MDA-MB-468 and MDA-MB-231) byhuman macrophages with and without anti-LILRB1 antibody (and in thepresence or absence of other antibodies, e.g., Hu5F9-G4 which is ananti-CD47 antibody). Bars from left to right are: IgG₄, Hu5F9-G4,Cetuximab, Trastuzumab, Panitumumab, Cetuximab+Hu5F9-G4,Trastuzumab+Hu5F9-G4, and Panitumumab+Hu5F9-G4.

FIG. 18. Data showing that CD47 and MHC class I signaling axes areindependent anti-phagocytic signals.

DETAILED DESCRIPTION

Methods and compositions are provided for inducing phagocytosis of atarget cell, treating an individual having cancer, treating anindividual having an intracellular pathogen infection (e.g., a chronicinfection), and/or reducing the number of inflicted cells (e.g., cancercells, cells infected with an intracellular pathogen, etc.) in anindividual. Methods and compositions are also provided for predictingwhether an individual is resistant (or susceptible) to treatment with ananti-CD47/SIRPA agent. In some cases, the subject methods andcompositions include an anti-MHC Class I/LILRB1 agent. In some cases,the subject methods and compositions include an anti-MHC Class I/LILRB1agent and an agent that opsonizes a target cell (e.g., co-administrationof an anti-MHC Class I/LILRB1 agent and an agent that opsonizes a targetcell). In some cases, the subject methods and compositions include ananti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPA agent (e.g.,co-administration of an anti-MHC Class I/LILRB1 agent and ananti-CD47/SIRPA agent). Kits are also provided for practicing themethods of the disclosure.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

In the description that follows, a number of terms conventionally usedin the field are utilized. In order to provide a clear and consistentunderstanding of the specification and claims, and the scope to be givento such terms, the following definitions are provided.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an.alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.In some embodiments, the mammal is human.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas cancer cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc.

The term “biological sample” encompasses a clinical sample, and alsoincludes tissue obtained by surgical resection, tissue obtained bybiopsy, cells in culture, cell supernatants, cell lysates, tissuesamples, organs, bone marrow, blood, plasma, serum, aspirate, and thelike. A “biological sample” includes a sample comprising target cellsand/or normal control cells, or is suspected of comprising such cells.The definition includes biological fluids derived therefrom (e.g.,cancerous cell, infected cell, etc.), e.g., a sample comprisingpolynucleotides and/or polypeptides that is obtained from such cells(e.g., a cell lysate or other cell extract comprising polynucleotidesand/or polypeptides). A biological sample comprising an inflicted cell(e.g., cancer cell, an infected cell, etc.) from a patient can alsoinclude non-inflicted cells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of cancer, the determination thatan individual is resistant or susceptible to treatment with an anti-CD47reagent, and the like.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of disease progression (e.g., cancer-attributable death orprogression, progression of an infection, etc.), including recurrence,metastatic spread of cancer, and drug resistance (e.g., resistance totreatment with an anti-CD47/SIRPA agent).

The term “prediction” is used herein to refer to the act of foretellingor estimating, based on observation, experience, or scientificreasoning. In one example, a physician may predict the likelihood that apatient will survive, following surgical removal of a primary tumorand/or chemotherapy for a certain period of time without cancerrecurrence. As another example, one may predict the likelihood that anindividual is resistant (or susceptible) to treatment with ananti-CD47/SIRPA agent (e.g. determine whether an individual is likely tobe resistant to treatment with an anti-CD47/SIRPA agent, or instead islikely to respond to treatment with an anti-CD47/SIRPA agent, i.e.,likely to be susceptible to treatment with an anti-CD47/SIRPA agent). Asyet another example, one may predict the likelihood that an individualis susceptible to treatment with an anti-CD47/SIRPA agent (e.g.determine whether an individual is likely to be susceptible to treatmentwith an anti-CD47/SIRPA agent).

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides/epitopes). In some embodiments,the affinity of one molecule for another molecule to which itspecifically binds is characterized by a KD (dissociation constant) of10⁻⁵ M or less (e.g., 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less,10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, 10⁻¹² M or less, 10⁻¹³M or less, 10⁻¹⁴ M or less, 10⁻¹⁵ M or less, or 10⁻¹⁶ M or less).“Affinity” refers to the strength of binding, increased binding affinitybeing correlated with a lower K_(D).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member). Examples of specificbinding members include, but are not limited to: agents thatspecifically bind MHC Class I (e.g., classical MHC Class I), LILRB1,CD47, and/or SIRPA (i.e., anti-MHC Class I/LILRB1 agents,anti-CD47/SIRPA agents), or that otherwise block the interaction betweenMHC Class I and LILRB1; and/or the interaction between CD47 and SIRPA.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments (e.g., Fab fragments) solong as they exhibit the desired biological activity. “Antibodies” (Abs)and “immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light- and heavy-chain variable domains (Clothia et al., J.Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.U.S.A. 82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a b-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the b-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Digestion of antibodies (e.g., with enzymes such as papain, Ficin, andthe like) produces two identical antigen-binding fragments, called “Fab”fragments, each with a single antigen-binding site, and a residual “Fc”fragment, whose name reflects its ability to crystallize readily. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species(scFv), one heavy- and one light-chain variable domain can be covalentlylinked by a flexible peptide linker such that the light and heavy chainscan associate in a “dimeric” structure analogous to that in a two-chainFv species. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called a, d, e, g, and m, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. Engineered variants of immunoglobulinsubclasses, including those that increase or decrease immune effectorfunctions, half-life, or serum-stability, are also encompassed by thisterminology.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)₂, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

Unless specifically indicated to the contrary, the term “conjugate” asdescribed and claimed herein is defined as a heterogeneous moleculeformed by the covalent attachment of one or more antibody fragment(s) toone or more polymer molecule(s), where the heterogeneous molecule iswater soluble, i.e. soluble in physiological fluids such as blood, andwherein the heterogeneous molecule is free of any structured aggregate.A conjugate of interest is PEG. In the context of the foregoingdefinition, the term “structured aggregate” refers to (1) any aggregateof molecules in aqueous solution having a spheroid or spheroid shellstructure, such that the heterogeneous molecule is not in a micelle orother emulsion structure, and is not anchored to a lipid bilayer,vesicle or liposome; and (2) any aggregate of molecules in solid orinsolubilized form, such as a chromatography bead matrix, that does notrelease the heterogeneous molecule into solution upon contact with anaqueous phase. Accordingly, the term “conjugate” as defined hereinencompasses the aforementioned heterogeneous molecule in a precipitate,sediment, bioerodible matrix or other solid capable of releasing theheterogeneous molecule into aqueous solution upon hydration of thesolid.

As used in this disclosure, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly, e.g., to asubject anti-MHC ClassI/LILRB1 agent and/or anti-CD47/SIRPA agent. Thelabel may itself be detectable by itself (directly detectable label)(e.g., radioisotope labels or fluorescent labels) or, or the label canbe indirectly detectable, e.g., in the case of an enzymatic label, theenzyme may catalyze a chemical alteration of a substrate compound orcomposition and the product of the reaction is detectable.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arefour main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

Compositions

The present disclosure provides compositions for inducing phagocytosisof a target cell, treating an individual having cancer, treating anindividual having an intracellular pathogen infection (e.g., a chronicinfection), reducing the number of inflicted cells (e.g., cancer cells,cells infected with an intracellular pathogen, etc.) in an individual,and/or predicting whether an individual is resistant (or susceptible) totreatment with an anti-CD47/SIRPA agent. In some cases, the subjectcompositions include an anti-MHC Class I/LILRB1 agent. In some cases,the subject compositions include an anti-MHC Class I/LILRB1 agent and ananti-CD47/SIRPA agent.

Anti-MHC Class I/LILRB1 agent. A major histocompatibility complex (MHC)Class I complex is made of human leukocyte antigen (HLA) alpha chainsand a beta-2-macroglobulin protein (B2M) that assemble to form acomplex. HLA alpha chains in humans include the alpha chains HLA-A,HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, and HLA-L. The HLA-A, HLA-B,and HLA-C alpha chains are referred to herein as classical HLA alphachains. Non-classical MHC Class I HLA alpha chains include HLA-E, HLA-F,HLA-G, HLA-K, and HLA-L. Thus, the term “classical MHC Class I” refersto MHC Class I complexes that include HLA-A, HLA-B, and/or HLA-C (and donot include HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L; and the term “MHCClass I” refers to any MHC Class I complex.

An MHC Class I complex on a first cell (e.g., a cancer cell, an infectedcell) can bind to (and activate) LILRB1 on a second cell (e.g., aphagocytic cell, e.g., a macrophage) and thereby inhibit phagocytosis ofthe first cell by the second cell. “Leukocyte immunoglobulin-likereceptor, subfamily B (with TM and ITIM domains), member 1” (LILRB1) isa member of the subfamily B class of leukocyte immunoglobulin-likereceptor (LIR) receptors, and contains an ectodomain (havingextracellular immunoglobulin domains), a transmembrane domain, and acytoplasmic domain (having immunoreceptor tyrosine-based inhibitorymotifs (ITIMs)). LILRB1 is expressed on immune cells where it binds toMHC class I molecules. When “activated,” the receptor transduces anegative signal that inhibits stimulation of an immune response in thecells on which it is expressed.

LILRB1 is also known as ILT2, ILT-2, CD85, CD85J, LIR1, LIR-1, and MIR7.The human LILRB1 protein exists as at least 6 different isoforms (setforth as SEQ ID NOs: 1-6):

LILRB1 (isoform 1) (SEQ ID NO: 1)MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDENPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH LILRB1 (isoform 2)(SEQ ID NO: 2)MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDENPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRQSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH LILRB1 (isoform 3)(SEQ ID NO: 3)MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDENPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH LILRB1 (isoform 4)(SEQ ID NO: 4)MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDENPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRQSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH LILRB1 (isoform 5)(SEQ ID NO: 5)MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDENPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH LILRB1 (isoform 6) (SEQ ID NO: 6)MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDENPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSAGPEDQPLTPTGSDPQSGE

As used herein, the term “anti-MHC Class I/LILRB1 agent” refers to anyagent that reduces the binding of MHC Class I (e.g., on a target cell)to LILRB1 (e.g., on a phagocytic cell). In some cases, an anti-MHC ClassI/LILRB1 agent binds to (specifically binds) MHC Class I (e.g.,classical MHC Class I) (e.g., binds to the fully assembled complex). Insome cases, an anti-MHC Class I/LILRB1 agent binds to (specificallybinds) one or more components of MHC Class I (e.g., one or more MHCClass I alpha chains, one or more classical MHC Class I alpha chains,and/or B2M), thereby reducing the formation of MHC Class I, whichresults in reduced binding of MHC Class I to LILRB1. In some cases, ananti-MHC Class I/LILRB1 agent (e.g. an anti-LILRB1 antibody, a solubleMHC class I complex) binds to (specifically binds) LILRB1.

Thus, in some embodiments, a suitable anti-MHC Class I/LILRB1 agent(e.g. an anti-MHC Class I antibody, a LILRB1 peptide, etc.) specificallybinds MHC Class I and reduces the binding of MHC Class Ito LILRB1. Insome embodiments, a suitable anti-MHC Class I/LILRB1 agent (e.g. ananti-MHC Class I antibody, a LILRB1 peptide, etc.) specifically bindsclassical MHC Class I (MHC Class I complexes that do not include anon-classical HLA alpha chain) and reduces the binding of classical MHCClass Ito LILRB1. In some embodiments, a suitable anti-MHC ClassI/LILRB1 agent (e.g. an anti-LILRB1 antibody, a soluble MHC class Icomplex) specifically binds LILRB1.

In some cases, an anti-MHC ClassI/LILRB1 agent (e.g., in any of themethods or compositions of the disclosure) is an antibody (e.g.,anti-LLRB1 antibody, anti-MHC Class I antibody) and in some cases it isa humanized antibody (e.g., can be an IgG₄ isotype humanized antibody,e.g., an IgG₄ isotype antibody having a mutation in the hinge regionsuch as the S241 P mutation that reduces heterogeneity sometimes foundin chimeric mouse/human IgG₄ antibodies)(e.g., see Angal et al., MolImmunol. 1993 January; 30(1):105-8).

Examples of suitable anti-MHC Class I/LILRB1 agents (MHC Class I bindingagents, e.g., classical MHC Class I binding agents, as well as LILRB1binding agents) include, but are not limited to: (i) anti-MHC Class Iantibodies (e.g., antibodies that bind to MHC Class I, antibodies thatbind to classical MHC Class I, which include an HLA-A, HLA-B, and/orHLA-C alpha chain); and (ii) LILRB1 peptides, including withoutlimitation soluble LILRB1 polypeptides that bind to MHC Class I, e.g., apolypeptide comprising an extracellular portion (ectodomain) of LILRB1,a high affinity LILRB1 polypeptide, etc.; (iii) anti-LILRB1 antibodies;and (iv) soluble MHC class I complexes that bind to LILRB1. Smallmolecule compounds that inhibit the binding of MHC Class I (e.g.,classical MHC Class I) with LILRB1 are also considered to be anti-MHCClass I/LILRB1 agents.

(i) and (ii) above are examples of MHC Class I binding agents (e.g.,classical MHC Class I binding agents); while (iii) and (iv) above areexamples of LILRB1 binding agents.

Anti-MHC Class I/LILRB1 agents (e.g., LILRB1 binding agents) do notactivate/stimulate LILRB1 (e.g., in the LILRB1-expressing phagocyticcell). In some cases, anti-MHC Class I/LILRB1 agents (e.g., LILRB1binding agents) do not activate/stimulate LILRB1 to an amount wheresignaling via LILRB1is stimulated on phagocytic cells, therebyinhibiting phagocytosis by the phagocytic cells. In other words, in somecases, a suitable anti-MHC Class I/LILRB1 agent that binds LILRB1 canstimulate some level of signaling via LILRB1 on phagocytic cells (i.e.,some level of signaling may be tolerated), as long as the level ofsignaling is not enough to inhibit phagocytosis.

(i) Anti-MHC Class I antibodies. In some embodiments, a subject anti-MHCClass

I/LILRB1 agent is an antibody (an anti-MHC Class I antibody) thatspecifically binds MHC class I (e.g., MCH Class I alpha chains HLA-A,HLA-B, and/or HLA-C) and reduces the interaction between MHC Class I onone cell (e.g., an infected cell) and LILRB1 on another cell (e.g., aphagocytic cell). The term “anti-MHC Class I antibody” as used hereinencompasses molecules that include the binding region of an anti-MHCClass I antibody, e.g., a molecule that includes the CDRs of an anti-MHCClass I antibody such as a Fab fragment (see definition of the term“antibody” above). Suitable anti-MHC Class I antibodies include fullyhuman, humanized or chimeric versions of such antibodies. For example,humanized antibodies are useful for in vivo applications in humans dueto their low antigenicity. Similarly caninized, felinized, etc.antibodies are useful for applications in dogs, cats, and other speciesrespectively. Antibodies of interest include humanized antibodies, orcaninized, felinized, equinized, bovinized, porcinized, etc.,antibodies, and variants thereof. In some cases, an anti-MHC Class Iantibody can be an anti-MHC Class I antibody that does not activate MHCClass I upon binding. Also envisioned are single chain antibodiesderived from camelids, single chain antibodies derived from shark,engineered fibronectin domain-containing proteins, knottin peptides, andDARPins; and fluorophore-conjugated versions of each of these reagents.

Examples of monoclonal anti-MHC Class I antibodies can include, but arenot limited to clones: W6/32, EP1395Y, OX18, ERMP42, MEM-E/02, 2G₅,F21-2, 41.17, OX-27, and 3D12HLA-E. In some embodiments, therefore, thedisclosure provides humanized versions of the above described monoclonalantibodies (e.g., those antibodies that recognize human MHC Class I,e.g., human classical MHC Class I). For any of the described anti-MHCClass I antibodies, the antibody can be a humanized antibody, a bindingfragment thereof (e.g., a Fab fragment), or any permutation having theantigen binding domain (or, e.g., the CDRs of the antigen bindingdomain). (See definition of “antibody” above).

In general, humanized antibodies are made by substituting amino acids inthe framework regions of a parent non-human antibody to produce amodified antibody that is less immunogenic in a human than the parentnon-human antibody. Antibodies can be humanized using a variety oftechniques known in the art including, for example, CDR-grafting (EP239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnickaet al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Incertain embodiments, framework substitutions are identified by modelingof the interactions of the CDR and framework residues to identifyframework residues important for antigen binding and sequence comparisonto identify unusual framework residues at particular positions (see,e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)).Additional methods for humanizing antibodies contemplated herein aredescribed in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403;5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCTpublications WO 98/45331 and WO 98/45332.

(ii) Soluble LILRB1 peptide. A soluble LILRB1 peptide comprises theportion of LILRB1 that is sufficient to bind MHC Class I at arecognizable affinity (e.g., on a target cell such as a cancer cell),which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Asubject soluble LILRB1 peptide does not include the transmembrane domainof LILRB1. A soluble LILRB1 peptide can comprise one or more of theextracellular immunoglobulin domains of LILRB1 (e.g., a soluble LILRB1peptide can include all or a portion of the soluble portion of theLILRB1 ectodomain). A soluble LILRB1 peptide reduces (e.g., blocks,prevents, etc.) the interaction between LILRB1 and MHC Class I.

Suitable soluble LILRB1 peptides include any peptide comprising variantor naturally existing LILRB1 sequences (e.g., extracellular domainsequences or extracellular domain variants) that can specifically bindLILRB1 and inhibit the interaction between MHC Class I (e.g., classicalMHC Class I) and LILRB1 without stimulating enough LILRB1 activity toinhibit phagocytosis. In some embodiments, a subject soluble LILRB1peptide comprises the extracellular domain of LILRB1, including a signalpeptide (e.g., the signal peptide of LILRB1). In some embodiments, thesignal peptide amino acid sequence may be substituted with a signalpeptide amino acid sequence that is derived from another polypeptide(e.g., for example, an immunoglobulin or CTLA4). For example, unlikefull-length LILRB1, which is a cell surface polypeptide that traversesthe outer cell membrane, soluble LILRB1 peptides are secreted;accordingly, a soluble LILRB1 peptide may include a heterologous signalpeptide that is normally associated with a polypeptide that is secretedfrom a cell. As described herein, signal peptides are not exposed on thecell surface of a secreted or transmembrane protein because either thesignal peptide is cleaved during translocation of the protein or thesignal peptide remains anchored in the outer cell membrane (such apeptide is also called a signal anchor). Without wishing to be bound bytheory, the signal peptide sequence of LILRB1 is believed to be cleavedfrom the precursor LILRB1 polypeptide in vivo. In some embodiments, asubject LILRB1 peptide comprises all or a portion of the extracellulardomain of LILRB1, but does not include a signal peptide.

Suitable soluble LILRB1 peptides include LILRB1 peptides havingextracellular domain mutations (variants) relative to a wild type LILRB1sequence. In some cases, a subject soluble LILRB1 peptide includes anamino acid sequence having 65% or more (e.g., 70% or more, 75% or more,80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% ormore, 99% or more, 99.5% or more, 99.8% or more, or 100%) amino acidsequence identity with the extracellular portion of an amino acidsequence set forth in any of SEQ ID NOs: 1-6, which variants retain thecapability to bind to LILRB1 without stimulating LILRB1 signaling enoughto inhibit phagocytosis by an LILRB1-expressing phagocytic cell. Assaysfor measuring whether a given peptide fulfills the above criteria arereadily available to one of ordinary skill in the art and any convenientassay can be used. In some embodiments, a subject anti-MHC ClassI/LILRB1 agent is a “high affinity LILRB1 peptide”, which includesLILRB1-derived polypeptides and analogs thereof. High affinity LILRB1peptides are variants of an above described LILRB1 peptide that compriseat least one amino acid change relative to the wild-type LILRB1sequence, where the amino acid change increases the affinity of theLILRB1 peptide for binding to MHC Class I (e.g., classical MHC ClassI)(e.g., by decreasing the off-rate by at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 500-fold, ormore).

In some embodiments, an LILRB1 peptide (e.g., a high affinity LILRB1peptide) is a fusion protein, e.g., fused in frame with a secondpolypeptide. In some embodiments, the second polypeptide is capable ofincreasing the size of the fusion protein, e.g., so that the fusionprotein will not be cleared from the circulation rapidly. In someembodiments, the second polypeptide is part or whole of animmunoglobulin Fc region. The Fc region can aid in phagocytosis byproviding an “eat me” signal, which enhances the block of the “don't eatme” signal provided by the LILRB1 peptide. In other embodiments, thesecond polypeptide is any suitable polypeptide that is substantiallysimilar to Fc, e.g., providing increased size, multimerization domains,and/or additional binding or interaction with Ig molecules.

(iii) Anti-LILRB1 antibodies. In some embodiments, a subject anti-MHCClass I/LILRB1 agent is an antibody that specifically binds LILRB1(i.e., an anti-LILRB1 antibody) and reduces the interaction between MHCClass I on one cell (e.g., an infected cell) and LILRB1 on another cell(e.g., a phagocytic cell). Suitable anti-LILRB1 antibodies can bindLILRB1 without activating/stimulating signaling through LILRB1 enough toinhibit phagocytosis. Thus, a suitable anti-LILRB1 antibody specificallybinds LILRB1 (without activating/stimulating enough of a signalingresponse to inhibit phagocytosis) and blocks an interaction betweenLILRB1 and MHC Class I (e.g., classical MHC Class I). Suitableanti-LILRB1 antibodies include fully human, humanized or chimericversions of such antibodies. For example, humanized antibodies areuseful for in vivo applications in humans due to their low antigenicity.Similarly caninized, felinized, etc. antibodies are useful forapplications in dogs, cats, and other species respectively. Antibodiesof interest include humanized antibodies, or caninized, felinized,equinized, bovinized, porcinized, etc., antibodies, and variantsthereof.

Examples of monoclonal anti-LILRB1 antibodies can include, but are notlimited to clones: GHI/75, HP-F1, 3D3-1D12, and VMP55. In someembodiments, therefore, the disclosure provides humanized versions ofthe above described monoclonal antibodies (e.g., those antibodies thatrecognize human LILRB1). Also envisioned are single chain antibodiesderived from camelids, single chain antibodies derived from shark,engineered fibronectin domain-containing proteins, knottin peptides, andDARPins; and fluorophore-conjugated versions of each of these reagents.

In general, humanized antibodies are made by substituting amino acids inthe framework regions of a parent non-human antibody to produce amodified antibody that is less immunogenic in a human than the parentnon-human antibody. Antibodies can be humanized using a variety oftechniques known in the art including, for example, CDR-grafting (EP239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnickaet al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Incertain embodiments, framework substitutions are identified by modelingof the interactions of the CDR and framework residues to identifyframework residues important for antigen binding and sequence comparisonto identify unusual framework residues at particular positions (see,e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)).Additional methods for humanizing antibodies contemplated herein aredescribed in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403;5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCTpublications WO 98/45331 and WO 98/45332.

The inventors have sequenced the GHI/75 antibody and have identified theantigen binding site/domain (the light and heavy chain sequences):

Light chain (VJ region) of clone GHI/75(anti-LILRB1 antibody)(underlined are the CDRs according to IMGT):(SEQ ID NO: 7) DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATNLADGVPSRFSGSGSGTKFSFKISSLQAEDFV SYYCQQLYSTPFTFGSGTKLEIKCDR-L1: (SEQ ID NO: 8) QTIGTW CDR-L2: (SEQ ID NO: 9) AAT CDR-L3:(SEQ ID NO: 10) CQQLYSTPFT Heavy chain (VDJ region) of clone GHI/75(anti-LILRB1 antibody)(underlined are the CDRs according to IMGT):(SEQ ID NO: 11) EVILVESGGALVRPGGSLKLSCAASGFTFSSNAMSWVRQTPEKRLEWVATISNGGTFTYYPDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYYCARHGDGNYGDPLDYWGQGTTLTVSS CDR-H1: (SEQ ID NO: 12) FTFSSNACDR-H2: (SEQ ID NO: 13) ISNGGTFT CDR-H3: (SEQ ID NO: 14) CARHGDGNYGDPL

For any of the described anti-LILRB1 antibodies, the antibody can be ahumanized antibody, a binding fragment thereof (e.g., a Fab fragment),or any permutation having the antigen binding domain (or, e.g., the CDRsof the antigen binding domain). (See definition of “antibody” above).

A subject anti-LILRB1 antibody may include: (i) one or more (e.g., 2 ormore, 3 or more, 4 or more, or 5 or more) CDR sequences (e.g., those setforth in SEQ ID NOs: 8-10 and 12-14); (ii) a complete variable region(e.g., those set forth in SEQ ID NOs: 7 and 11); and/or (iii)single-chain variable fragments (e.g., that include any or all of thesequences set forth in SEQ ID NOs: 7-14). As is known in the art, avariable region sequence may be fused to any appropriate constant regionsequence.

In some embodiments a subject anti-LILRB1 antibody includes one moreCDRs (e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 6 CDRs) thatincludes an amino acid sequence set forth in SEQ ID NOs: 8-10 and 12-14.A subject anti-LILRB1 antibody can include a CDR sequence that differsby up to 6 amino acids (e.g., up to 5 amino acids, up to 4 amino acids,up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid) ascompared to a CDR amino acid sequence set forth in any of SEQ ID NOs:8-10 and 12-14.

In some cases, a subject anti-LILRB1 antibody includes one or more CDRs(e.g., 2 or more, 3 or more, 4 or more, 5 or more, or 6) having an aminoacid sequence that differs by up to 6 amino acids (e.g., up to 5 aminoacids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, orup to 1 amino acid) as compared to a CDR amino acid sequence set forthin any of SEQ ID NOs: 8-10 and 12-14. In some cases, a subjectanti-LILRB1 antibody includes two or more CDRs (e.g., 3 or more, 4 ormore, 5 or more, 6, or 6 or more) that have an amino acid sequence thatdiffers by up to 6 amino acids (e.g., up to 5 amino acids, up to 4 aminoacids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid)as compared to a CDR amino acid sequence set forth in any of SEQ ID NOs:8-10 and 12-14.

In some embodiments, a subject anti-LILRB1 antibody includes an aminoacid sequence that is 80% or more (e.g., 85% or more, 90% or more, 92%or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% ormore, or 100%) identical to a CDR amino acid sequence set forth in anyof SEQ ID NOs: 8-10 and 12-14. In some cases, a subject anti-LILRB1antibody includes a heavy chain having one or more (e.g., two or more,three or more, or 3) of the amino acid sequences set forth in SEQ IDNOs: 12-14. In some cases, a subject anti-LILRB1 antibody includes aheavy chain having all 3 of the amino acid sequences set forth in SEQ IDNOs: 12-14. In some cases, a subject anti-LILRB1 antibody includes alight chain having one or more (e.g., two or more, three or more, or 3)of the amino acid sequences set forth in SEQ ID NOs: 8-10. In somecases, a subject anti-LILRB1 antibody includes a light chain having all3 of the amino acid sequences set forth in SEQ ID NOs: 8-10.

In some cases, a subject anti-LILRB1 antibody includes a light chainhaving all 3 of the amino acid sequences set forth in SEQ ID NOs: 8-10,and a heavy chain having all 3 of the amino acid sequences set forth inSEQ ID NOs: 12-14.

In some cases, a subject anti-LILRB1 antibody includes a heavy chainhaving three CDRs, where CDR-H1 has the amino acid sequence set forth inSEQ ID NO: 12, CDR-H2 has the amino acid sequence set forth in SEQ IDNO: 13, and CDR-H3 has the amino acid sequence set forth in SEQ ID NO:14. In some cases, a subject anti-LILRB1 antibody includes a light chainhaving three CDRs, where CDR-L1 has the amino acid sequence set forth inSEQ ID NO: 8, CDR-L2 has the amino acid sequence set forth in SEQ ID NO:9, and CDR-L3 has the amino acid sequence set forth in SEQ ID NO: 10. Insome cases, a subject anti-LILRB1 antibody includes: (i) a heavy chainhaving three CDRs, where CDR-H1 has the amino acid sequence set forth inSEQ ID NO: 12, CDR-H2 has the amino acid sequence set forth in SEQ IDNO: 13, and CDR-H3 has the amino acid sequence set forth in SEQ ID NO:14; and (ii) a light chain having three CDRs, where CDR-L1 has the aminoacid sequence set forth in SEQ ID NO: 8, CDR-L2 has the amino acidsequence set forth in SEQ ID NO: 9, and CDR-L3 has the amino acidsequence set forth in SEQ ID NO: 10.

In some cases, a subject anti-LILRB1 antibody includes a heavy chainhaving an amino acid sequence as set forth in SEQ ID NO: 11. In somecases, a subject anti-LILRB1 antibody includes a light chain havingamino acid sequence as set forth in SEQ ID NO: 7. In some cases, asubject anti-LILRB1 antibody includes a heavy chain having an amino acidsequence as set forth in SEQ ID NO: 11; and a light chain having anamino acid sequence as set forth in SEQ ID NO: 7.

(iv) Soluble MHC Class I complex. In some embodiments, a subjectanti-MHC Class I/LILRB1 agent is a soluble MHC Class I complex (or anycomponent thereof) that specifically binds LILRB1 and reduces theinteraction between MHC Class I on one cell (e.g., an infected cell) andLILRB1 on another cell (e.g., a phagocytic cell). A suitable soluble MHCClass I complex can bind LILRB1 without activating or stimulatingsignaling through LILRB1 because activation of LILRB1 would inhibitphagocytosis. A suitable soluble MHC Class I polypeptide specificallybinds LILRB1 without activating/stimulating enough of a signalingresponse to inhibit phagocytosis.

Efficacy of an anti-MHC Class I/LILRB1 agent. The efficacy of a suitableanti-MHC Class I/LILRB1 agent can be assessed by assaying the agent. Asa non-limiting example of such an assay, target cells are incubated inthe presence or absence of the candidate agent, and phagocytosis of thetarget cells is measured (e.g., phagocytosis by macrophages). An agentfor use in the subject methods (an anti-MHC Class I/LILRB1 agent) willup-regulate phagocytosis by at least 10% (e.g., at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 120%, at least 140%, at least160%, at least 180%, at least 200%, or at least 300%) compared tophagocytosis in the absence of the candidate agent. Any convenientphagocytosis assay can be used. As a non-limiting example of aphagocytosis assay, see the Examples below.

In some cases, the assay can be conducted in the presence of a knownphagocytosis inducing agent (e.g., an anti-CD47/SIRPA agent). In somecases, in the presence of a known phagocytosis inducing agent (e.g., ananti-CD47/SIRPA agent), an anti-MHC Class I/LILRB1 agent willup-regulate regulate phagocytosis by at least 10% (e.g., at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 120%, at least 140%, atleast 160%, at least 180%, at least 200%, or at least 300%) compared tophagocytosis in the absence of the phagocytosis inducing agent. In somecases, in the presence of a known phagocytosis inducing agent (e.g., ananti-CD47/SIRPA agent), an anti-MHC Class I/LILRB1 agent willup-regulate regulate phagocytosis by at least 10% (e.g., at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 120%, at least 140%, atleast 160%, at least 180%, at least 200%, or at least 300%) compared tophagocytosis in the absence of the candidate agent.

Similarly, an in vitro assay that measures tyrosine phosphorylation ofLILRB1 can be used (e.g., as an alternative or in addition to aphagocytosis assay). In some cases, a suitable candidate agent will showa decrease in phosphorylation by at least 5% (e.g., at least 10%, atleast 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or 100%) comparedto phosphorylation observed in absence of the candidate agent. In somecases, in the presence of a known phagocytosis inducing agent (e.g., ananti-CD47/SIRPA agent), a suitable candidate anti-MHC Class I/LILRB1agent will show a decrease in phosphorylation by at least 5% (e.g., atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or100%) compared to phosphorylation in the absence of the phagocytosisinducing agent. In some cases, in the presence of a known phagocytosisinducing agent (e.g., an anti-CD47/SIRPA agent), a suitable candidateanti-MHC Class I/LILRB1 agent will show a decrease in phosphorylation byat least 5% (e.g., at least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or 100%) compared to phosphorylation in the absenceof the candidate agent.

Anti-CD47/SIRPA agent. As used herein, the term “anti-CD47/SIRPA agent”refers to any agent that reduces the binding of CD47, e.g., on a targetcell, to SIRPA (also known as SIRPα), e.g., on a phagocytic cell.Non-limiting examples of suitable anti-CD47/SIRPA agents include SIRPAreagents, including without limitation high affinity SIRPA polypeptides;anti-SIRPA antibodies; soluble CD47 polypeptides; and anti-CD47antibodies or antibody fragments. In some embodiments, a suitableanti-CD47/SIRPA agent (e.g. an anti-CD47 antibody, a SIRPA reagent,etc.) specifically binds CD47 to reduce the binding of CD47 to SIRPA. Insome embodiments, a suitable anti-CD47/SIRPA agent (e.g., an anti-SIRPAantibody, a soluble CD47 polypeptide, etc.) specifically binds SIRPA toreduce the binding of CD47 to SIRPA. A suitable anti-CD47/SIRPA agentthat binds SIRPA does not activate SIRPA (e.g., in the SIRPA-expressingphagocytic cell). The efficacy of a suitable anti-CD47/SIRPA agent canbe assessed by assaying the agent (further described below). As anon-limiting example of such an assay, target cells are incubated in thepresence or absence of the candidate agent, and phagocytosis of thetarget cells is measured (e.g., phagocytosis by macrophages). An agentfor use in the subject methods (an anti-CD47/SIRPA agent) willup-regulate phagocytosis by at least 10% (e.g., at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 120%, at least 140%, at least160%, at least 180%, at least 200%, or at least 300%) compared tophagocytosis in the absence of the candidate agent. Any convenientphagocytosis assay can be used. As a non-limiting example of aphagocytosis assay, see the Examples below. Similarly, an in vitro assaythat measures tyrosine phosphorylation of SIRPA can be used (e.g., as analternative or in addition to a phagocytosis assay). A suitablecandidate agent will show a decrease in phosphorylation by at least 5%(e.g., at least 10%, at least 15%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or 100%) compared to phosphorylation observed in absence of thecandidate agent.

In some embodiments, the anti-CD47/SIRPA agent does not activate CD47upon binding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) may occur (Manna and Frazier, Cancer Research,64, 1026-1036, Feb. 1, 2004). Thus, in some embodiments, theanti-CD47/SIRPA agent does not directly induce cell death of aCD47-expressing cell.

Some pathogens (e.g., pox viruses, Myxoma virus, Deerpox virus, swinepoxvirus, goatpox virus, sheeppox virus, etc.) express a CD47-analog (i.e.,a CD47 mimic) (e.g., the M128L protein) that acts as a virulence factorto enable infection (Cameron et al., Virology. 2005 Jun. 20;337(1):55-67), and some pathogens induce the expression of endogenousCD47 in the host cell. Cells infected with a pathogen that expresses aCD47-analog may therefore express the pathogen-provided CD47 analogeither exclusively or in combination with endogenous CD47. Thismechanism allows the pathogen to increase CD47 expression (viaexpression of the CD47 analog) in the infected cell with or withoutincreasing the level of endogenous CD47. In some embodiments, ananti-CD47/SIRPA agent (e.g., anti-CD47 antibody, a SIRPA reagent, aSIRPA antibody, a soluble CD47 polypeptide, etc.) can reduce the bindingof a CD47 analog (i.e., a CD47 mimic) to SIRPA. In some cases, asuitable anti-CD47/SIRPA agent (e.g., a SIRPA reagent, an anti-CD47antibody, etc.) can bind a CD47 analog (i.e., a CD47 mimic) to reducethe binding of the CD47 analog to SIRPA. In some cases, a suitableanti-CD47/SIRPA agent (e.g., an anti-SIRPA antibody, a soluble CD47polypeptide, etc.) can bind to SIRPA. A suitable anti-CD47/SIRPA agentthat binds SIRPA does not activate SIRPA (e.g., in the SIRPA-expressingphagocytic cell). An anti-CD47/SIRPA agent can be used in any of themethods provided herein when the pathogen is a pathogen that provides aCD47 analog. In other words the term “CD47,” as used herein, encompassesCD47 as well as CD47 analogs (i.e., CD47 mimics).

SIRPA reagent. A SIRPA reagent comprises the portion of SIRPA that issufficient to bind CD47 at a recognizable affinity, which normally liesbetween the signal sequence and the transmembrane domain, or a fragmentthereof that retains the binding activity. A suitable SIRPA reagentreduces (e.g., blocks, prevents, etc.) the interaction between thenative proteins SIRPA and CD47. The SIRPA reagent will usually compriseat least the d1 domain of SIRPA. In some embodiments, a SIRPA reagent isa fusion protein, e.g., fused in frame with a second polypeptide. Insome embodiments, the second polypeptide is capable of increasing thesize of the fusion protein, e.g., so that the fusion protein will not becleared from the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPA reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules.

In some embodiments, a subject anti-CD47/SIRPA agent is a “high affinitySIRPA reagent”, which includes SIRPA-derived polypeptides and analogsthereof. High affinity SIRPA reagents are described in internationalapplication PCT/U.S. Ser. No. 13/21,937, which is hereby specificallyincorporated by reference. High affinity SIRPA reagents are variants ofthe native SIRPA protein. In some embodiments, a high affinity SIRPAreagent is soluble, where the polypeptide lacks the SIRPA transmembranedomain and comprises at least one amino acid change relative to thewild-type SIRPA sequence, and wherein the amino acid change increasesthe affinity of the SIRPA polypeptide binding to CD47, for example bydecreasing the off-rate by at least 10-fold, at least 20-fold, at least50-fold, at least 100-fold, at least 500-fold, or more.

A high affinity SIRPA reagent comprises the portion of SIRPA that issufficient to bind CD47 at a recognizable affinity, e.g., high affinity,which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Thehigh affinity SIRPA reagent will usually comprise at least the d1 domainof SIRPA with modified amino acid residues to increase affinity. In someembodiments, a SIRPA variant of the present invention is a fusionprotein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. The Fcregion aids in phagocytosis by providing an “eat me” signal, whichenhances the block of the “don't eat me” signal provided by the highaffinity SIRPA reagent. In other embodiments, the second polypeptide isany suitable polypeptide that is substantially similar to Fc, e.g.,providing increased size, multimerization domains, and/or additionalbinding or interaction with Ig molecules. The amino acid changes thatprovide for increased affinity are localized in the d1 domain, and thushigh affinity SIRPA reagents comprise a d1 domain of human SIRPA, withat least one amino acid change relative to the wild-type sequence withinthe d1 domain. Such a high affinity SIRPA reagent optionally comprisesadditional amino acid sequences, for example antibody Fc sequences;portions of the wild-type human SIRPA protein other than the d1 domain,including without limitation residues 150 to 374 of the native proteinor fragments thereof, usually fragments contiguous with the d1 domain;and the like. High affinity SIRPA reagents may be monomeric ormultimeric, i.e. dimer, trimer, tetramer, etc. An example of ahigh-affinity SIRPA reagent is known as CV1 (an engineered proteinmonomer).

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47/SIRPAagent is an antibody that specifically binds CD47 (i.e., an anti-CD47antibody) and reduces the interaction between CD47 on one cell (e.g., aninfected cell) and SIRPA on another cell (e.g., a phagocytic cell). Insome embodiments, a suitable anti-CD47 antibody does not activate CD47upon binding. Non-limiting examples of suitable antibodies includeclones B6H12, 5F9, 8B6, and C3 (for example as described inInternational Patent Publication WO 2011/143624, herein specificallyincorporated by reference). Suitable anti-CD47 antibodies include fullyhuman, humanized or chimeric versions of such antibodies. Humanizedantibodies (e.g., hu5F9-G4) are especially useful for in vivoapplications in humans due to their low antigenicity. Similarlycaninized, felinized, etc. antibodies are especially useful forapplications in dogs, cats, and other species respectively. Antibodiesof interest include humanized antibodies, or caninized, felinized,equinized, bovinized, porcinized, etc., antibodies, and variantsthereof.

Anti-SIRPA antibodies. In some embodiments, a subject anti-CD47/SIRPAagent is an antibody that specifically binds SIRPA (i.e., an anti-SIRPAantibody) and reduces the interaction between CD47 on one cell (e.g., aninfected cell) and SIRPA on another cell (e.g., a phagocytic cell).Suitable anti-SIRPA antibodies can bind SIRPA without activating orstimulating signaling through SIRPA because activation of SIRPA wouldinhibit phagocytosis. Instead, suitable anti-SIRPA antibodies facilitatethe preferential phagocytosis of inflicted cells over normal cells.Those cells that express higher levels of CD47 (e.g., infected cells)relative to other cells (non-infected cells) will be preferentiallyphagocytosed. Thus, a suitable anti-SIRPA antibody specifically bindsSIRPA (without activating/stimulating enough of a signaling response toinhibit phagocytosis) and blocks an interaction between SIRPA and CD47.Suitable anti-SIRPA antibodies include fully human, humanized orchimeric versions of such antibodies. Humanized antibodies areespecially useful for in vivo applications in humans due to their lowantigenicity. Similarly caninized, felinized, etc. antibodies areespecially useful for applications in dogs, cats, and other speciesrespectively. Antibodies of interest include humanized antibodies, orcaninized, felinized, equinized, bovinized, porcinized, etc.,antibodies, and variants thereof.

Soluble CD47 polypeptides. In some embodiments, a subjectanti-CD47/SIRPA agent is a soluble CD47 polypeptide that specificallybinds SIRPA and reduces the interaction between CD47 on one cell (e.g.,an infected cell) and SIRPA on another cell (e.g., a phagocytic cell). Asuitable soluble CD47 polypeptide can bind SIRPA without activating orstimulating signaling through SIRPA because activation of SIRPA wouldinhibit phagocytosis. Instead, suitable soluble CD47 polypeptidesfacilitate the preferential phagocytosis of infected cells overnon-infected cells. Those cells that express higher levels of CD47(e.g., infected cells) relative to normal, non-target cells (normalcells) will be preferentially phagocytosed. Thus, a suitable solubleCD47 polypeptide specifically binds SIRPA without activating/stimulatingenough of a signaling response to inhibit phagocytosis.

In some cases, a suitable soluble CD47 polypeptide can be a fusionprotein (for example as structurally described in US Patent PublicationUS20100239579, herein specifically incorporated by reference). However,only fusion proteins that do not activate/stimulate SIRPA are suitablefor the methods provided herein. Suitable soluble CD47 polypeptides alsoinclude any peptide or peptide fragment comprising variant or naturallyexisting CD47 sequences (e.g., extracellular domain sequences orextracellular domain variants) that can specifically bind SIRPA andinhibit the interaction between CD47 and SIRPA without stimulatingenough SIRPA activity to inhibit phagocytosis.

In certain embodiments, soluble CD47 polypeptide comprises theextracellular domain of CD47, including the signal peptide. Soluble CD47polypeptides also include CD47 extracellular domain variants thatcomprise an amino acid sequence at least 65%-75%, 75%-80%, 80-85%,85%-90%, or 95%-99% (or any percent identity not specifically enumeratedbetween 65% to 100%), which variants retain the capability to bind toSIRPA without stimulating SIRPA signaling.

In certain embodiments, the signal peptide amino acid sequence may besubstituted with a signal peptide amino acid sequence that is derivedfrom another polypeptide (e.g., for example, an immunoglobulin orCTLA4). For example, unlike full-length CD47, which is a cell surfacepolypeptide that traverses the outer cell membrane, the soluble CD47polypeptides are secreted; accordingly, a polynucleotide encoding asoluble CD47 polypeptide may include a nucleotide sequence encoding asignal peptide that is associated with a polypeptide that is normallysecreted from a cell.

In other embodiments, the soluble CD47 polypeptide comprises anextracellular domain of CD47 that lacks the signal peptide. As describedherein, signal peptides are not exposed on the cell surface of asecreted or transmembrane protein because either the signal peptide iscleaved during translocation of the protein or the signal peptideremains anchored in the outer cell membrane (such a peptide is alsocalled a signal anchor). The signal peptide sequence of CD47 is believedto be cleaved from the precursor CD47 polypeptide in vivo.

In other embodiments, a soluble CD47 polypeptide comprises a CD47extracellular domain variant. Such a soluble CD47 polypeptide retainsthe capability to bind to SIRPA without stimulating SIRPA signaling. TheCD47 extracellular domain variant may have an amino acid sequence thatis at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical(which includes any percent identity between any one of the describedranges) to the extracellular domain of wild type CD47.

The above described agents can be prepared in a variety of ways. Forexample, an anti-MHC Class I/LILRB1 agent and/or anti-CD47/SIRPA agentcan be prepared (together or separately): as a dosage unit, with apharmaceutically acceptable excipient, with pharmaceutically acceptablesalts and esters, etc. Compositions can be provided as pharmaceuticalcompositions.

Pharmaceutical Compositions. Suitable anti-MHC Class I/LILRB1 agentsand/or anti-CD47/SIRPA agents can be provided in pharmaceuticalcompositions suitable for therapeutic use, e.g. for human treatment. Insome embodiments, pharmaceutical compositions of the present inventioninclude one or more therapeutic entities of the present disclosure(e.g., an anti-MHC ClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent)and include a pharmaceutically acceptable carrier, a pharmaceuticallyacceptable salt, a pharmaceutically acceptable excipient, and/or estersor solvates thereof. In some embodiments, the use of an anti-MHC ClassI/LILRB1 agent and/or anti-CD47/SIRPA agent includes use in combinationwith another therapeutic agent (e.g., another anti-infection agent oranother anti-cancer agent). Therapeutic formulations comprising ananti-MHC Class I/LILRB1 agent and/or an anti-CD47/SIRPA agent can beprepared by mixing the agent(s) having the desired degree of purity witha physiologically acceptable carrier, a pharmaceutically acceptablesalt, an excipient, and/or a stabilizer (Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980)) (e.g., in the form oflyophilized formulations or aqueous solutions). A composition having ananti-MHC Class I/LILRB1 agent and/or an anti-CD47/SIRPA agent can beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

Methods

Methods are provided for inducing phagocytosis of a target cell,treating an individual having cancer, treating an individual having anintracellular pathogen infection (e.g., a chronic infection), reducingthe number of inflicted cells (e.g., cancer cells, cells infected withan intracellular pathogen, etc.) in an individual, and/or predictingwhether an individual is resistant (or susceptible) to treatment with ananti-CD47/SIRPA agent. In some cases, the subject methods include theuse of an anti-MHC Class I/LILRB1 agent and an agent that opsonizes atarget cell (e.g., co-administration of an anti-MHC Class I/LILRB1 agentand an agent that opsonizes a target cell). In some cases, the subjectmethods include the use of an anti-MHC Class I/LILRB1 agent and ananti-CD47/SIRPA agent (e.g., co-administration of an anti-MHC ClassI/LILRB1 agent and an anti-CD47/SIRPA agent). In some cases, the subjectmethods include the use of an anti-MHC Class I/LILRB1 agent, ananti-CD47/SIRPA agent, and an agent that opsonizes a target cell (e.g.,co-administration of an anti-MHC Class I/LILRB1 agent, ananti-CD47/SIRPA agent, and an agent that opsonizes a target cell). Insome cases an anti-CD47/SIRPA agent is an agent that opsonizes a targetcell (e.g., when the anti-CD47/SIRPA agent is an anti-CD47 antibodyhaving an Fc region).

The compositions described above can find use in the methods describedherein.

In some cases, a subject method is a method of inducing phagocytosis ofa target cell.

The term “target cell” as used herein refers to a cell (e.g., inflictedcells such as cancer cells, infected cells, etc.) that is targeted forphagocytosis by a phagocytic cell. In some cases, a target cell isresistant to treatment with an anti-CD47/SIRPA agent. For example, someinflicted cells (e.g., cancer cells) do not express MHC Class I and suchcells are susceptible to an anti-CD47/SIRPA agent. When a target cellthat is susceptible to an anti-CD47/SIRPA agent is contacted with aphagocytic cell in the presence of an anti-CD47/SIRPA agent, the targetcell can be engulfed (e.g., phagocytosed) by the phagocytic cell.

However, some inflicted cells (e.g., cancer cells) do express MHC ClassI and such cells can be resistant to an anti-CD47/SIRPA agent. When atarget cell that is resistant to an anti-CD47/SIRPA agent is contactedwith a phagocytic cell (e.g., a macrophage) in the presence of ananti-CD47/SIRPA agent, the target cell is less likely to be engulfed(e.g., phagocytosed) by the phagocytic cell (see the working examplesbelow). In some embodiments, a target cell (e.g., a target cell that isresistant to an anti-CD47/SIRPA agent) is contacted with a phagocyticcell (e.g., a macrophage) in the presence of an anti-MHC Class I/LILRB1agent and an anti-CD47/SIRPA agent. When a target cell that is resistantto an anti-CD47/SIRPA agent (e.g., the resistant target cell expressesMHC Class I) is contacted with a phagocytic cell (e.g., a macrophage) inthe presence of an anti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPAagent, the phagocytic cell can engulf (e.g., phagocytose) the targetcell. Contacting a target cell with a phagocytic cell (e.g., amacrophage) in the presence of an anti-MHC Class I/LILRB1 agent and ananti-CD47/SIRPA agent encompasses scenarios where the target cell iscontacted with the anti-MHC Class I/LILRB1 agent and the anti-CD47/SIRPAagent at the same time (i.e, both agents are present at the same time),and scenarios where the target cell is contacted with one of the agentsprior to the other agent (in either order)(e.g., one of the agents ispresent first, and the other agent is later added, either in thepresence or absence of the first agent).

Contacting a target cell with a phagocytic cell (e.g., a macrophage) inthe present of an anti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPAagent can occur in vitro or in vivo. For example, in some cases, atarget cell (e.g., a cancer cell from an individual, a cancer cell of animmortalized cell line, an infected cell from an individual, an infectedcell of a cell line, and the like) is cultured in vitro with aphagocytic cell, an anti-MHC Class I/LILRB1 agent, and ananti-CD47/SIRPA agent.

In some cases, after the phagocytic cell engulfs the target cell, thephagocytic cell is introduced into an individual (e.g., the individualfrom whom the target cell was taken). In some cases, the phagocytic cellis a cell from an individual (e.g., the same individual from whom thetarget cell was taken) and the phagocytic cell is re-introduce into theindividual after the phagocytic cell engulfs the target cell. When thetarget cell and/or the phagocytic cell is from an individual that isbeing treated, the method can be referred to as an ex vivo method. Insome cases, a method of inducing phagocytosis of a target cell, wherethe method includes contacting the target cell with a phagocytic cell(e.g., a macrophage) in the presence of an anti-MHC Class I/LILRB1 agentand an anti-CD47/SIRPA agent, can occur in vivo. In such cases, theanti-MHC Class I/LILRB1 agent and the anti-CD47/SIRPA agent can beadministered to an individual (e.g., an individual having cancer, achronic infection, etc.) and the contact of the target cell with thephagocytic cell will happen in vivo, without further input from the oneperforming the method. As such, in some cases, a method of inducingphagocytosis of a target cell can encompass a method that includesadministering to an individual an anti-MHC Class I/LILRB1 agent and ananti-CD47/SIRPA agent.

A target cell may be a cell that is “inflicted”, where the term“inflicted” is used herein to refer to a subject with symptoms, anillness, or a disease that can be treated with an anti-MHC ClassI/LILRB1 agent and an anti-CD47/SIRPA agent. An “inflicted” subject canhave cancer, can harbor an infection (e.g., a chronic infection), andother hyper-proliferative conditions, for example sclerosis, fibrosis,and the like, etc. “Inflicted cells” may be those cells that cause thesymptoms, illness, or disease. As non-limiting examples, the inflictedcells of an inflicted patient can be cancer cells, infected cells, andthe like. One indication that an illness or disease can be treated withan anti-CD47/SIRPA agent is that the involved cells (i.e., the inflictedcells, e.g., the cancerous cells, the infected cells, etc.) express anincreased level of CD47 compared to normal cells of the same cell type.One indication that an illness or disease can be treated with ananti-MHC Class I/LILRB1 agent is that the involved cells (i.e., theinflicted cells, e.g., the cancerous cells, the infected cells, etc.)express MHC Class I (e.g., classical MHC Class I). In some cases, anindication that an illness or disease can be treated with an anti-MHCClass I/LILRB1 agent and an anti-CD47/SIRPA agent is that the involvedcells (i.e., the inflicted cells, e.g., the cancerous cells, theinfected cells, etc.) express an increased level of CD47 compared tonormal cells of the same cell type, and express MHC Class I (e.g.,classical MHC Class I).

In some cases, a subject method is a method of treating an individualhaving cancer and/or having an intracellular pathogen infection (e.g., achronic infection). An effective treatment will reduce the number ofinflicted cells (e.g., cancer cells, cells infected with anintracellular pathogen, etc.) in an individual (e.g., via increasingphagocytosis of the target cells). As such, in some cases, a subjectmethod is a method of reducing the number of inflicted cells (e.g.,cancer cells, cells infected with an intracellular pathogen, etc.) in anindividual.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom(s) thereofand/or may be therapeutic in terms of a partial or completestabilization or cure for a disease and/or adverse effect attributableto the disease. The term “treatment” encompasses any treatment of adisease in a mammal, particularly a human, and includes: (a) preventingthe disease and/or symptom(s) from occurring in a subject who may bepredisposed to the disease or symptom but has not yet been diagnosed ashaving it; (b) inhibiting the disease and/or symptom(s), i.e., arrestingtheir development; or (c) relieving the disease symptom(s), i.e.,causing regression of the disease and/or symptom(s). Those in need oftreatment include those already inflicted (e.g., those with cancer,those with an infection, those with an immune disorder, etc.) as well asthose in which prevention is desired (e.g., those with increasedsusceptibility to cancer, those with an increased likelihood ofinfection, those suspected of having cancer, those suspected ofharboring an infection, etc.).

A therapeutic treatment is one in which the subject is inflicted priorto administration and a prophylactic treatment is one in which thesubject is not inflicted prior to administration. In some embodiments,the subject has an increased likelihood of becoming inflicted or issuspected of being inflicted prior to treatment. In some embodiments,the subject is suspected of having an increased likelihood of becominginflicted.

Examples of symptoms, illnesses, and/or diseases that can be treatedwith an anti-MHC Class I/LILRB1 agent (e.g. in combination with ananti-CD47/SIRPA agent) include, but are not limited to cancer (any formof cancer, including but not limited to: carcinomas, soft tissue tumors,sarcomas, teratomas, melanomas, leukemias, lymphomas, brain cancers,solid tumors, mesothelioma (MSTO), etc.); infection from anintracellular pathogen (e.g., chronic infection); and immunologicaldiseases or disorders (e.g., an inflammatory disease)(e.g., multiplesclerosis, arthritis, and the like)(e.g., for immunosuppressivetherapy).

As used herein “cancer” includes any form of cancer, including but notlimited to solid tumor cancers (e.g., lung, prostate, breast, bladder,colon, ovarian, pancreas, kidney, liver, glioblastoma, medulloblastoma,leiomyosarcoma, head & neck squamous cell carcinomas, melanomas,neuroendocrine; etc.) and liquid cancers (e.g., hematological cancers);carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas;leukemias; lymphomas; and brain cancers, including minimal residualdisease, and including both primary and metastatic tumors. Any cancer isa suitable cancer to be treated by the subject methods and compositions.

Carcinomas are malignancies that originate in the epithelial tissues.Epithelial cells cover the external surface of the body, line theinternal cavities, and form the lining of glandular tissues. Examples ofcarcinomas include, but are not limited to: adenocarcinoma (cancer thatbegins in glandular (secretory) cells), e.g., cancers of the breast,pancreas, lung, prostate, and colon can be adenocarcinomas;adrenocortical carcinoma; hepatocellular carcinoma; renal cellcarcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma;carcinoma of the breast; basal cell carcinoma; squamous cell carcinoma;transitional cell carcinoma; colon carcinoma; nasopharyngeal carcinoma;multilocular cystic renal cell carcinoma; oat cell carcinoma; large celllung carcinoma; small cell lung carcinoma; non-small cell lungcarcinoma; and the like. Carcinomas may be found in prostrate, pancreas,colon, brain (usually as secondary metastases), lung, breast, skin, etc.

Soft tissue tumors are a highly diverse group of rare tumors that arederived from connective tissue. Examples of soft tissue tumors include,but are not limited to: alveolar soft part sarcoma; angiomatoid fibroushistiocytoma; chondromyoxid fibroma; skeletal chondrosarcoma;extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplasticsmall round-cell tumor; dermatofibrosarcoma protuberans; endometrialstromal tumor; Ewing's sarcoma; fibromatosis (Desmoid); fibrosarcoma,infantile; gastrointestinal stromal tumor; bone giant cell tumor;tenosynovial giant cell tumor; inflammatory myofibroblastic tumor;uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindlecell or pleomorphic lipoma; atypical lipoma; chondroid lipoma;well-differentiated liposarcoma; myxoid/round cell liposarcoma;pleomorphic liposarcoma; myxoid malignant fibrous histiocytoma;high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignantperipheral nerve sheath tumor; mesothelioma; neuroblastoma;osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolarrhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignantschwannoma; synovial sarcoma; Evan's tumor; nodular fasciitis;desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcomaprotuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma;tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis(PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovialsarcoma; malignant peripheral nerve sheath tumor; neurofibroma; andpleomorphic adenoma of soft tissue; and neoplasias derived fromfibroblasts, myofibroblasts, histiocytes, vascular cells/endothelialcells and nerve sheath cells.

A sarcoma is a rare type of cancer that arises in cells of mesenchymalorigin, e.g., in bone or in the soft tissues of the body, includingcartilage, fat, muscle, blood vessels, fibrous tissue, or otherconnective or supportive tissue. Different types of sarcoma are based onwhere the cancer forms. For example, osteosarcoma forms in bone,liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examplesof sarcomas include, but are not limited to: askin's tumor; sarcomabotryoides; chondrosarcoma; ewing's sarcoma; malignanthemangioendothelioma; malignant schwannoma; osteosarcoma; and softtissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma;cystosarcoma phyllodesdermatofibrosarcoma protuberans (DFSP); desmoidtumor; desmoplastic small round cell tumor; epithelioid sarcoma;extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma;gastrointestinal stromal tumor (GIST); hemangiopericytoma;hemangiosarcoma (more commonly referred to as “angiosarcoma”); kaposi'ssarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignantperipheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovialsarcoma; undifferentiated pleomorphic sarcoma, and the like).

A teratoma is a type of germ cell tumor that may contain severaldifferent types of tissue (e.g., can include tissues derived from anyand/or all of the three germ layers: endoderm, mesoderm, and ectoderm),including for example, hair, muscle, and bone. Teratomas occur mostoften in the ovaries in women, the testicles in men, and the tailbone inchildren.

Melanoma is a form of cancer that begins in melanocytes (cells that makethe pigment melanin). It may begin in a mole (skin melanoma), but canalso begin in other pigmented tissues, such as in the eye or in theintestines.

Leukemias are cancers that start in blood-forming tissue, such as thebone marrow, and causes large numbers of abnormal blood cells to beproduced and enter the bloodstream. For example, leukemias can originatein bone marrow-derived cells that normally mature in the bloodstream.Leukemias are named for how quickly the disease develops and progresses(e.g., acute versus chronic) and for the type of white blood cell thatis affected (e.g., myeloid versus lymphoid). Myeloid leukemias are alsocalled myelogenous or myeloblastic leukemias. Lymphoid leukemias arealso called lymphoblastic or lymphocytic leukemia. Lymphoid leukemiacells may collect in the lymph nodes, which can become swollen. Examplesof leukemias include, but are not limited to: Acute myeloid leukemia(AML), Acute lymphoblastic leukemia (ALL), Chronic myeloid leukemia(CML), and Chronic lymphocytic leukemia (CLL).

Lymphomas are cancers that begin in cells of the immune system. Forexample, lymphomas can originate in bone marrow-derived cells thatnormally mature in the lymphatic system. There are two basic categoriesof lymphomas. One kind is Hodgkin lymphoma (HL), which is marked by thepresence of a type of cell called the Reed-Sternberg cell. There arecurrently 6 recognized types of HL. Examples of Hodgkin lymphomasinclude: nodular sclerosis classical Hodgkin lymphoma (CHL), mixedcellularity CHL, lymphocyte-depletion CHL, lymphocyte-rich CHL, andnodular lymphocyte predominant HL.

The other category of lymphoma is non-Hodgkin lymphomas (NHL), whichincludes a large, diverse group of cancers of immune system cells.Non-Hodgkin lymphomas can be further divided into cancers that have anindolent (slow-growing) course and those that have an aggressive(fast-growing) course. There are currently 61 recognized types of NHL.Examples of non-Hodgkin lymphomas include, but are not limited to:AIDS-related Lymphomas, anaplastic large-cell lymphoma,angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt'slymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma),chronic lymphocytic leukemia/small lymphocytic lymphoma, cutaneousT-Cell lymphoma, diffuse large B-Cell lymphoma, enteropathy-type T-Celllymphoma, follicular lymphoma, hepatosplenic gamma-delta T-Celllymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle celllymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatriclymphoma, peripheral T-Cell lymphomas, primary central nervous systemlymphoma, transformed lymphomas, treatment-related T-Cell lymphomas, andWaldenstrom's macroglobulinemia.

Brain cancers include any cancer of the brain tissues. Examples of braincancers include, but are not limited to: gliomas (e.g., glioblastomas,astrocytomas, oligodendrogliomas, ependymomas, and the like),meningiomas, pituitary adenomas, vestibular schwannomas, primitiveneuroectodermal tumors (medulloblastomas), etc.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body.

As used herein, the term “infection” refers to any state in at least onecell of an organism (i.e., a subject) is infected by an infectious agent(e.g., a subject has an intracellular pathogen infection, e.g., achronic intracellular pathogen infection). As used herein, the term“infectious agent” refers to a foreign biological entity (i.e. apathogen) (e.g., one that induces increased CD47 expression in at leastone cell of the infected organism). For example, infectious agentsinclude, but are not limited to bacteria, viruses, protozoans, andfungi. Intracellular pathogens are also of interest. Infectious diseasesare disorders caused by infectious agents. Some infectious agents causeno recognizable symptoms or disease under certain conditions, but havethe potential to cause symptoms or disease under changed conditions. Thesubject methods can be used in the treatment of chronic pathogeninfections, for example including but not limited to viral infections,e.g. retrovirus, lentivirus, hepadna virus, herpes viruses, pox viruses,human papilloma viruses, etc.; intracellular bacterial infections, e.g.Mycobacterium, Chlamydophila, Ehrlichia, Rickettsia, Brucella,Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella,Yersinia sp, Helicobacter pylori etc.; and intracellular protozoanpathogens, e.g. Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasmasp., Leishmania sp., etc.

Infectious diseases that can be treated using a subject anti-MHC ClassI/LILRB1 agent and/or anti-CD47/SIRPA agent include but are not limitedto: HIV, Influenza, Herpes, Giardia, Malaria, Leishmania, the pathogenicinfection by the virus Hepatitis (A, B, & C), herpes virus (e.g., VZV,HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus,influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus,cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measlesvirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, denguevirus, papillomavirus, molluscum virus, poliovirus, rabies virus, JCvirus and arboviral encephalitis virus, pathogenic infection by thebacteria chlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumonococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, E. coli, legionella, diphtheria,salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague,leptospirosis, and Lyme's disease bacteria, pathogenic infection by thefungi Candida (albicans, krusei, glabrata, tropicalis, etc.),Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), GenusMucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis andHistoplasma capsulatum, and pathogenic infection by the parasitesEntamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoebasp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosomacruzi, Leishmania donovani, Toxoplasma gondi, and/or Nippostrongylusbrasiliensis.

In some embodiments the infliction is a chronic infection, i.e. aninfection that is not cleared by the host immune system within a periodof up to 1 week, 2 weeks, etc. In some cases, chronic infections involveintegration of pathogen genetic elements into the host genome, e.g.retroviruses, lentiviruses, Hepatitis B virus, etc. In other cases,chronic infections, for example certain intracellular bacteria orprotozoan pathogens, result from a pathogen cell residing within a hostcell. Additionally, in some embodiments, the infection is in a latentstage, as with herpes viruses or human papilloma viruses.

An infection treated with the methods of the invention generallyinvolves a pathogen with at least a portion of its life-cycle within ahost cell, i.e. an intracellular phase. The methods of the inventionprovide for a more effective removal of infected cells by the phagocyticcells of the host organism, relative to phagocytosis in the absence oftreatment, and thus are directed to the intracellular phase of thepathogen life cycle.

The terms “co-administration”, “co-administer”, and “in combinationwith” include the administration of two or more therapeutic agents(e.g., an anti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPA agent)either simultaneously, concurrently or sequentially within no specifictime limits. In one embodiment, the agents are present in the cell or inthe subject's body at the same time or exert their biological ortherapeutic effect at the same time. In one embodiment, the therapeuticagents are in the same composition or unit dosage form. In otherembodiments, the therapeutic agents are in separate compositions or unitdosage forms. In certain embodiments, a first agent can be administeredprior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second therapeutic agent.

In some cases, a subject an anti-MHC Class I/LILRB1 agent and/or ananti-CD47/SIRPA agent (e.g., formulated as a pharmaceutical composition)is co-administered with a cancer therapeutic drug, therapeutic drug totreat an infection, or tumor-directed antibody. Such administration mayinvolve concurrent (i.e. at the same time), prior, or subsequentadministration of the drug/antibody with respect to the administrationof an agent or agents of the disclosure. A person of ordinary skill inthe art would have no difficulty determining the appropriate timing,sequence and dosages of administration for particular drugs andcompositions of the present disclosure.

In some embodiments, treatment is accomplished by administering acombination (co-administration) of a subject anti-MHC Class I/LILRB1agent (e.g., with or without an anti-CD47/SIRPA agent) with anotheragent (e.g., an immune stimulant, an agent to treat chronic infection, acytotoxic agent, an anti-cancer agent, etc.). One example class ofcytotoxic agents that can be used are chemotherapeutic agents. Exemplarychemotherapeutic agents include, but are not limited to, aldesleukin,altretamine, amifostine, asparaginase, bleomycin, capecitabine,carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim,fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea,idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole,levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide,mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel(Taxol™), pilocarpine, prochloroperazine, rituximab, saproin, tamoxifen,taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristineand vinorelbine tartrate.

An anti-MHC ClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent need notbe, but is optionally formulated with one or more agents that potentiateactivity, or that otherwise increase the therapeutic effect. These aregenerally used in the same dosages and with administration routes asused herein or from 1 to 99% of the heretofore employed dosages. In someembodiments, treatment is accomplished by administering a combination(co-administration) of a subject anti-MHC Class I/LILRB1 agent and anagent that opsonizes a target cell. In some embodiments, treatment isaccomplished by administering a combination (co-administration) of asubject anti-MHC Class I/LILRB1 agent, an agent that opsonizes a targetcell, and an anti-CD47/SIRPA agent. In some embodiments, treatment isaccomplished by administering a combination (co-administration) of asubject anti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPA agent.Thus, also envisioned herein are compositions (and methods that use thecompositions) that include: (a) an anti-MHC ClassI/LILRB1 agent; and (b)at least one of: (i) an agent that opsonizes the target cell, and (ii)an anti-CD47/SIRPA agent. In some cases, that agent that opsonizes thetarget cell is Rituximab. In some cases, that agent that opsonizes thetarget cell is Cetuximab.

An “agent that opsonizes a target cell” (an “opsonizing agent”) is anyagent that can bind to a target cell (e.g., a cancer cell, a cellharboring an intracellular pathogen, etc.) and opsonize the target cell.For example, any antibody that can bind to a target cell (as definedherein), where the antibody has an FC region, is considered to be anagent that opsonizes a target cell. In some cases, the agent thatopsonizes a target cell is an antibody, other than an anti-CD47antibody, that binds to a target cell (e.g., an anti-tumor antibody, ananti-cancer antibody, an anti-infection antibody, and the like).

For example antibodies selective for tumor cell markers, radiation,surgery, and/or hormone deprivation, see Kwon et al., Proc. Natl. Acad.Sci U.S.A., 96: 15074-9, 1999. Angiogenesis inhibitors can also becombined with the methods of the invention. A number of antibodies arecurrently in clinical use for the treatment of cancer, and others are invarying stages of clinical development. For example, there are a numberof antigens and corresponding monoclonal antibodies for the treatment ofB cell malignancies. One target antigen is CD20. Rituximab is a chimericunconjugated monoclonal antibody directed at the CD20 antigen. CD20 hasan important functional role in B cell activation, proliferation, anddifferentiation. The CD52 antigen is targeted by the monoclonal antibodyalemtuzumab, which is indicated for treatment of chronic lymphocyticleukemia. CD22 is targeted by a number of antibodies, and has recentlydemonstrated efficacy combined with toxin in chemotherapy-resistanthairy cell leukemia. Two new monoclonal antibodies targeting CD20,tositumomab and ibritumomab, have been submitted to the Food and DrugAdministration (FDA). These antibodies are conjugated withradioisotopes. Alemtuzumab (Campath) is used in the treatment of chroniclymphocytic leukemia; Gemtuzumab (Mylotarg) finds use in the treatmentof acute myelogenous leukemia; Ibritumomab (Zevalin) finds use in thetreatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use inthe treatment of colon cancer.

Monoclonal antibodies useful in the methods of the invention that havebeen used in solid tumors include, without limitation, edrecolomab andtrastuzumab (herceptin). Edrecolomab targets the 17-1A antigen seen incolon and rectal cancer, and has been approved for use in Europe forthese indications. Trastuzumab targets the HER-2/neu antigen. Thisantigen is seen on 25% to 35% of breast cancers. Cetuximab (Erbitux) isalso of interest for use in the methods of the invention. The antibodybinds to the EGF receptor (EGFR), and has been used in the treatment ofsolid tumors including colon cancer and squamous cell carcinoma of thehead and neck (SCCHN).

A subject anti-MHC Class I/LILRB1 agent can be combined (with or withoutan anti-CD47/SIRPA agent) any of the above mentioned antibodies (agentsthat opsonize a target cell). Thus, in some cases, a subject anti-MHCClass I/LILRB1 agent, e.g., an agent that specifically binds classicalMHC Class I (e.g., an anti-MHC Class I antibody, a LILRB1 peptide)and/or an agent that specifically binds LILRB1 (e.g. an anti-LILRB1antibody, a soluble MHC class I complex), is used in a combinationtherapy (is co-administered) with one or more cell-specific antibodiesselective for tumor cell markers. in some cases, a subject anti-MHCClass I/LILRB1 agent, e.g., an agent that specifically binds classicalMHC Class I (e.g., an anti-MHC Class I antibody, a LILRB1 peptide)and/or an agent that specifically binds LILRB1 (e.g. an anti-LILRB1antibody, a soluble MHC class I complex), is used in a combinationtherapy (is co-administered) with an anti-CD47/SIRPA agent and one ormore cell-specific antibodies selective for tumor cell markers.

In some cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with one or more of:cetuximab (binds EGFR), panitumumab (binds EGFR), rituximab (bindsCD20), trastuzumab (binds HER2), pertuzumab (binds HER2), alemtuzumab(binds CD52), brentuximab (binds CD30), tositumomab, ibritumomab,gemtuzumab, ibritumomab, and edrecolomab (binds 17-1A).

In some cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with ananti-CD47/SIRPA agent and one or more of: cetuximab (binds EGFR),panitumumab (binds EGFR), rituximab (binds CD20), trastuzumab (bindsHER2), pertuzumab (binds HER2), alemtuzumab (binds CD52), brentuximab(binds CD30), tositumomab, ibritumomab, gemtuzumab, ibritumomab, andedrecolomab (binds 17-1A).

In some cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with one or moreagents that specifically bind one or more of: CD19, CD20, CD22, CD24,CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123,CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, andHAVCR2 (TIM3).

In some cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with ananti-CD47/SIRPA agent and one or more agents that specifically bind oneor more of: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52,CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR,17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

In some cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with any convenientimmunomodulatory agent (e.g., an anti-CTLA4 antibody, an anti-PD-1antibody, an anti-PD-L1 antibody, a CD40 agonist, a 4-1BB modulator(e.g., a 41BB-agonist), and the like).

Insome cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with ananti-CD47/SIRPA agent and any convenient immunomodulatory agent (e.g.,an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, aCD40 agonist, a 4-1BB modulator (e.g., a 41BB-agonist), and the like).

In some cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with an inhibitor ofBTLA and/or CD160.

Insome cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with ananti-CD47/SIRPA agent and an inhibitor of BTLA and/or CD160.

In some cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with an inhibitor ofTIM3 and/or CEACAM1.

Insome cases, a subject anti-MHC Class I/LILRB1 agent, e.g., an agentthat specifically binds classical MHC Class I (e.g., an anti-MHC Class Iantibody, a LILRB1 peptide) and/or an agent that specifically bindsLILRB1 (e.g. an anti-LILRB1 antibody, a soluble MHC class I complex), isused in a combination therapy (is co-administered) with ananti-CD47/SIRPA agent and an inhibitor of TIM3 and/or CEACAM1.

Treatment may also be combined with other active agents, such asantibiotics, cytokines, anti-viral agents, etc. Classes of antibioticsinclude penicillins, e.g. penicillin G, penicillin V, methicillin,oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins incombination with β-lactamase inhibitors, cephalosporins, e.g. cefaclor,cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams;aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins;sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin;trimethoprim; vancomycin; etc. Cytokines may also be included, e.g.interferon γ, tumor necrosis factor α, interleukin 12, etc. Antiviralagents, e.g. acyclovir, gancyclovir, etc., may also be used intreatment.

A “therapeutically effective dose” or “therapeutic dose” is an amountsufficient to effect desired clinical results (i.e., achieve therapeuticefficacy). A therapeutically effective dose can be administered in oneor more administrations. For purposes of this disclosure, atherapeutically effective dose of an anti-MHC Class I/LILRB1 agentand/or an anti-CD47/SIRPA agent is an amount that is sufficient topalliate, ameliorate, stabilize, reverse, prevent, slow or delay theprogression of the disease state (e.g., cancer or chronic infection) byincreasing phagocytosis of a target cell (e.g., a target cell). Thus, atherapeutically effective dose of an anti-MHC Class I/LILRB1 agentand/or an anti-CD47/SIRPA agent reduces the binding of (i) MHC on antarget cell, to LILRB1 on a phagocytic cell; and/or (ii) CD47 on antarget cell, to SIRPA on a phagocytic cell; at an effective dose forincreasing the phagocytosis of the target cell.

In some embodiments, a therapeutically effective dose leads to sustainedserum levels of an anti-MHC Class I/LILRB1 agent and/or ananti-CD47/SIRPA agent (e.g., an anti-MHC anti-body and/or an anti-CD47antibody) of 40 pg/ml or more (e.g, 50 ug/ml or more, 60 ug/ml or more,75 ug/ml or more, 100 ug/ml or more, 125 ug/ml or more, or 150 ug/ml ormore) for each agent. In some embodiments, a therapeutically effectivedose leads to sustained serum levels of an anti-MHC Class I/LILRB1 agentand/or an anti-CD47/SIRPA agent (e.g., an anti-MHC anti-body and/or ananti-CD47 antibody) that range from 40 pg/ml to 300 ug/ml (e.g, from 40ug/ml to 250 ug/ml, from 40 ug/ml to 200 ug/ml, from 40 ug/ml to 150ug/ml, from 40 ug/ml to 100 ug/ml, from 50 ug/ml to 300 ug/ml, from 50ug/ml to 250 ug/ml, from 50 ug/ml to 200 ug/ml, from 50 ug/ml to 150ug/ml, from 75 ug/ml to 300 ug/ml, from 75 ug/ml to 250 ug/ml, from 75ug/ml to 200 ug/ml, from 75 ug/ml to 150 ug/ml, from 100 ug/ml to 300ug/ml, from 100 ug/ml to 250 ug/ml, or from 100 ug/ml to 200 ug/ml) foreach agent. In some embodiments, a therapeutically effective dose fortreating solid tumors leads to sustained serum levels of an anti-MHCClass I/LILRB1 agent and/or an anti-CD47/SIRPA agent (e.g., an anti-MHCanti-body and/or an anti-CD47 antibody) of 100 pg/ml or more (e.g.,sustained serum levels that range from 100 ug/ml to 200 ug/ml) for eachagent. In some embodiments, a therapeutically effective dose fortreating non-solid tumors (e.g., acute myeloid leukemia (AML)) leads tosustained serum levels of an anti-MHC Class I/LILRB1 agent and/or ananti-CD47/SIRPA agent (e.g., an anti-MHC anti-body and/or an anti-CD47antibody) of 50 pg/ml or more (e.g., sustained serum levels of 75 pg/mlor more; or sustained serum levels that range from 50 ug/ml to 150ug/ml) for each agent.

Accordingly, a single therapeutically effective dose or a series oftherapeutically effective doses would be able to achieve and maintain aserum level of an anti-MHC Class I/LILRB1 agent and/or ananti-CD47/SIRPA agent. A therapeutically effective dose of an anti-MHCClass I/LILRB1 agent and/or an anti-CD47/SIRPA agent can depend on thespecific agent used, but is usually 8 mg/kg body weight or more (e.g., 8mg/kg or more, 10 mg/kg or more, 15 mg/kg or more, 20 mg/kg or more, 25mg/kg or more, 30 mg/kg or more, 35 mg/kg or more, or 40 mg/kg or more)for each agent, or from 10 mg/kg to 40 mg/kg (e.g., from 10 mg/kg to 35mg/kg, or from 10 mg/kg to 30 mg/kg) for each agent. The dose requiredto achieve and/or maintain a particular serum level is proportional tothe amount of time between doses and inversely proportional to thenumber of doses administered. Thus, as the frequency of dosingincreases, the required dose decreases. The optimization of dosingstrategies will be readily understood and practiced by one of ordinaryskill in the art. For all therapeutically effective doses listed above,when both an anti-MHC Class I/LILRB1agent and an anti-CD47/SIRPA agentare used, the dose for each agent can be independent from the otheragent. As an illustrative example (to illustrate the independence of thedoses), a therapeutic dose of the anti-MHC Class I/LILRB1agent may befrom 75 ug/ml to 250 ug/ml while a therapeutic dose of theanti-CD47/SIRPA agent may be from 40 ug/ml to 100 ug/ml.

Dosage and frequency may vary depending on the half-life of the anti-MHCClass I/LILRB1agent and/or anti-CD47/SIRPA agent in the patient. It willbe understood by one of skill in the art that such guidelines will beadjusted for the molecular weight of the active agent, e.g. in the useof antibody fragments, in the use of antibody conjugates, in the use ofanti-MHC Class I/LILRB1 agents, in the use of anti-CD47/SIRPA agents,etc. The dosage may also be varied for localized administration, e.g.intranasal, inhalation, etc., or for systemic administration, e.g. i.m.,i.p., i.v., and the like.

A sub-therapeutic dose is a dose (i.e., an amount) that is notsufficient to effect the desired clinical results when used in aparticular context. For example, a sub-therapeutic dose of ananti-CD47/SIRPA agent is an amount that is not sufficient to palliate,ameliorate, stabilize, reverse, prevent, slow or delay the progressionof the disease state (e.g., cancer, infection, inflammation, etc.).However, in some cases, when a sub-therapeutic dose of ananti-CD47/SIRPA agent is used in combination with (co-administered with)a subject anti-MHC Class I/LILRB1 agent, the dose can become atherapeutic dose. In other words, in some cases, a given dose of ananti-CD47/SIRPA agent can be sub-therapeutic when used in the absence ofa subject anti-MHC Class I/LILRB1 agent, but therapeutic when used inthe presence of an anti-MHC Class I/LILRB1 agent. Thus, in some cases,an anti-CD47/SIRPA agent can be co-administration with an anti-MHC ClassI/LILRB1 agent, where the dose of the anti-CD47/SIRPA agent wouldotherwise be sub-therapeutic (e.g., the dose of the anti-CD47/SIRPAagent would be sub-therapeutic when used in the absence of an anti-MHCClass I/LILRB1 agent. In some cases, it is desirable to use asub-therapeutic dose of an anti-CD47/SIRPA agent in combination with asubject anti-MHC Class I/LILRB1 agent. In some cases, a sub-therapeuticdose of an anti-CD47/SIRPA agent (sub-therapeutic when used in theabsence of an anti-MHC Class I/LILRB1 agent) is a therapeutic dose whenthe agent is used in combination (co-administered) with an anti-MHCClass I/LILRB1 agent.

While the use of a sub-therapeutic dose of an anti-CD47/SIRPA agent incombination with an anti-MHC ClassI/LILRB1 agent achieves a desiredoutcome (e.g., the combination is therapeutic), the dose is notconsidered to be a “therapeutic dose” because the sub-therapeutic dosedoes not effectively increase phagocytosis of a target cell and is notsufficient to palliate, ameliorate, stabilize, reverse, prevent, slow ordelay the progression of the disease state, unless used in combinationwith an anti-MHC ClassI/LILRB1 agent. A sub-therapeutic dose of ananti-CD47/SIRPA agent can depend on the specific agent used, but in somecases is less than 10 mg/kg.

In some cases, a sub-therapeutic dose of an anti-CD47/SIRPA agent isdesirable because some anti-CD47/SIRPA agents, when used at a highenough dose, can cause a reduction of red blood cells in an individualbeing treated. Thus, in some cases, co-administration with an anti-MHCClassI/LILRB1 agent allows for the anti-CD47/SIRPA agent to be used at adose that reduces the potential loss of red blood cells, but that wouldotherwise be considered a sub-therapeutic dose. As such, aco-administration may be able to circumvent the reduction of bloodcells.

An anti-MHC ClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent can beadministered by any suitable means, including topical, oral, parenteral,intrapulmonary, and intranasal. Parenteral infusions includeintramuscular, intravenous (bollus or slow drip), intraarterial,intraperitoneal, intrathecal or subcutaneous administration. An anti-MHCClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent can be administeredin any manner which is medically acceptable. This may includeinjections, by parenteral routes such as intravenous, intravascular,intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal,intraventricular, intraepidural, or others as well as oral, nasal,ophthalmic, rectal, or topical. Sustained release administration is alsospecifically included in the disclosure, by such means as depotinjections or erodible implants. Localized delivery is particularlycontemplated, by such means as delivery via a catheter to one or morearteries, such as the renal artery or a vessel supplying a localizedtumor.

-   As noted above, an anti-MHC ClassI/LILRB1 agent and/or an    anti-CD47/SIRPA agent can be formulated with an a pharmaceutically    acceptable carrier (one or more organic or inorganic ingredients,    natural or synthetic, with which a subject agent is combined to    facilitate its application). A suitable carrier includes sterile    saline although other aqueous and non-aqueous isotonic sterile    solutions and sterile suspensions known to be pharmaceutically    acceptable are known to those of ordinary skill in the art. An    “effective amount” refers to that amount which is capable of    ameliorating or delaying progression of the diseased, degenerative    or damaged condition. An effective amount can be determined on an    individual basis and will be based, in part, on consideration of the    symptoms to be treated and results sought. An effective amount can    be determined by one of ordinary skill in the art employing such    factors and using no more than routine experimentation.

An anti-MHC ClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent is oftenadministered as a pharmaceutical composition comprising an activetherapeutic agent and another pharmaceutically acceptable excipient. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

In some embodiments, pharmaceutical compositions can also include large,slowly metabolized macromolecules such as proteins, polysaccharides suchas chitosan, polylactic acids, polyglycolic acids and copolymers (suchas latex functionalized Sepharose™, agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins, peptides, and polysaccharides such as aminodextran, each ofwhich have multiple sites for the attachment of moieties. A carrier mayalso bear an anti-MHC ClassI/LILRB1 agent and/or an anti-CD47/SIRPAagent by non-covalent associations, such as non-covalent bonding or byencapsulation. The nature of the carrier can be either soluble orinsoluble for purposes of the invention. Those skilled in the art willknow of other suitable carriers for binding anti-MHC Class I/LILRB1agents and/or anti-CD47/SIRPA agents, or will be able to ascertain such,using routine experimentation.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Carriers and linkers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. A radionuclidechelate may be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, or metal oxide, radionuclide.

Radiographic moieties for use as imaging moieties in the presentinvention include compounds and chelates with relatively large atoms,such as gold, iridium, technetium, barium, thallium, iodine, and theirisotopes. It is preferred that less toxic radiographic imaging moieties,such as iodine or iodine isotopes, be utilized in the methods of theinvention. Such moieties may be conjugated to the anti-MHC ClassI/LILRB1agent and/or an anti-CD47/SIRPA agent through an acceptable chemicallinker or chelation carrier. Positron emitting moieties for use in thepresent invention include ¹⁸F, which can be easily conjugated by afluorination reaction with the anti-MHC ClassI/LILRB1 agent and/or ananti-CD47/SIRPA agent.

Compositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes,Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of thisinvention can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained or pulsatile release of the active ingredient. Thepharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the anti-MHC Class I/LILRB1 agents and/or anti-CD47/SIRPAagents can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀ (thedose lethal to 50% of the population) or the LD₁₀₀ (the dose lethal to100% of the population). The dose ratio between toxic and therapeuticeffect is the therapeutic index. The data obtained from these cellculture assays and animal studies can be used in further optimizingand/or defining a therapeutic dosage range and/or a sub-therapeuticdosage range (e.g., for use in humans). The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition.

In some cases, a method of inducing phagocytosis of a target cell,treating an individual having cancer, treating an individual having anintracellular pathogen infection (e.g., a chronic infection), and/orreducing the number of inflicted cells (e.g., cancer cells, cellsinfected with an intracellular pathogen, etc.) in an individual,includes, as described below, predicting whether an individual isresistant or susceptible to treatment with an anti-CD47/SIRPA agent.

Methods of Predicting

As discussed above, in some cases, a target cell (even one thatexpressed CD47) is relatively resistant to an anti-CD47/SIRPA agent,meaning that the target cell is less susceptible to phagocytois by aphagocytic cell (e.g., a macrophage), even when the target cell iscontacted by a phagocytic cell in the present of an anti-CD47/SIRPAagent. As such, in some cases, an individual (e.g, an individual havinginflicted cells, e.g., cancer cells and/or infected cells) can berelatively resistant to treatment with an anti-CD47/SIRPA agent. Also asdescribed above, and as described below in the working examples, theinventors have discovered that the level of expression of MHC Class I(e.g., classical MHC Class I) by an inflicted cell (eg., on the cellsurface) can be used to predict whether a target cell (and thereforewhether an individual) is resistant to treatment using ananti-CD47/SIRPA agent. In this context, resistance to treatment using ananti-CD47/SIRPA agent refers to treatment in the absence of a subjectanti-MHC Class I/LILRB1 agent, because the inventors have discoveredthat contacting a target cell (e.g., a target cell that is resistant totreatment with an anti-CD47/SIRPA agent) with an anti-MHC Class I/LILRB1agent can overcome the resistance.

The terms “resistance” and “resistant” (used herein when referring toresistance to an anti-CD47/SIRPA agent) is used herein to refer totarget cells that exhibit a decrease in the susceptibility tophagocytosis (in the present of an anti-CD47/SIRPA agent) compared toother cells. For example, while many cancer cells are negative for (orexpress low levels of) MHC Class I (e.g., classical MHC Class I), somecancer cells are positive for MHC Class I (e.g., classical MHC Class I).Target cells (e.g., cancer cells) can express MHC Class I (e.g.,classical MHC Class I) over a range of levels. For example, some targetcells express more MHC Class I (e.g., classical MHC Class I) thanothers, but still express less than normal cells. Some target cellsexpress normal levels of MHC Class I. The inventors have discovered thatthe level of MHC Class I expressed by a target cell correlates with itssusceptibility to phagocytosis by a phagocytic cell (e.g., amacrophage). Thus, when the term “resistance” or “resistant” is used, itdoes not necessarily mean that the cells cannot be phagocytosed, butdoes mean that the cells are not phagocytosed as efficiently as othercells (e.g., a smaller proportion of cells of a population of the cellscan be phagocytosed, e.g., over a given period of time, when compared toother cells).

In some embodiments, a target cell that is resistant to treatment withan anti-CD47/SIRPA agent exhibits a phagocytosis efficiency that is 95%or less (e.g., 90% or less, 85% or less, 80% or less, 75% or less, 70%or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% orless, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less,25% or less, 20% or less, 15% or less, or 10% or less) of thephagocytosis efficiency exhibited by a control cell (e.g., a controlpopulation of cells). In this context, a control cell can be any targetcell that expresses CD47 and is phagocytosed when contacted with: (i) aphagocytic cell (e.g., a macrophage), and (ii) an anti-CD47/SIRPA agent.For example, in some cases, a control cell in this context is a cancercell line known to be susceptible to an anti-CD47/SIRPA agent. Assays todetermine phagocytosis efficiency will be known to one of ordinary skillin the art and any convenient assay can be used. For example, see theworking examples below (e.g., see FIG. 1B). As such, an individual canbe predicted to be resistant to treatment with an anti-CD47/SIRPA agentwhen a target cell exhibits an MHC Class I expression level that isabove a particular threshold (which can be determined by comparing themeasured expression level to a level measured from a control cell thatis susceptible to treatment with an anti-CD47/SIRPA agent.

In some embodiments, a target cell (or an individual) is predicted to besusceptible to an anti-CD47/SIRPA agent when the target cell expresses95% or less (e.g., 90% or less, 85% or less, 80% or less, 75% or less,70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% orless, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less,25% or less, 20% or less, 15% or less, or 10% or less) MHC Class I asexpressed by a control cell (e.g., a normal cell). In some cases, atarget cell (or an individual) is predicted to be resistant to ananti-CD47/SIRPA agent when the target cell expresses 1.1-fold or more(e.g., 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold ormore, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold ormore, 2-fold or more, 2.1-fold or more, 2.5-fold or more, 3-fold ormore, 4-fold or more, 5-fold or more, etc.) MHC Class I compared to acontrol cell (e.g., an MHC Class I negative cell, a cell that expresseslow levels of MHC Class I but is known to be susceptible, and the like)or compared to a background value.

Methods of predicting whether target cells are (or an individual is)resistant or susceptible to treatment with an anti-CD47/SIRPA agentinclude the step of measuring the expression level of MajorHistocompatibility Complex (MHC) Class I in a biological sample of theindividual (e.g., where the biological sample comprises an inflictedcell such as a cancer cell or a cell harboring an intracellularpathogen), to produce a measured test value. The measured test value canthen be compared to a control value. In some cases, the value ismeasured for individual cells (e.g., using flow cytometry).

In some cases, when the measured test value is greater than or equal tothe control value, a prediction of resistance is made (and when themeasured test value is less than the control value, a prediction ofsusceptible is made). The control value can be a predetermined value orcan be a value that is measured around the same time that the test valueis measured. In some cases, the control value is a value of expressionwhich is known to be associated with a phenotype of resistance to ananti-CD47/SIRPA agent. As such, when the measured test value is equal toor greater than this value, a prediction of resistance can be made. Sucha control value (one that is known to be associated with a phenotype ofresistance to an anti-CD47/SIRPA agent) can be a value measured from aninflicted cell known to exhibit a phenotype of resistance.

In some cases, when the measured test value is greater than the controlvalue, a prediction of resistance is made (and when the measured testvalue is less than or equal to the control value, a prediction ofsusceptible is made). The control value can be a predetermined value orcan be a value that is measured at or around the same time that the testvalue is measured. In some cases, the control value is a valuerepresenting the background value of the measuring step (e.g., theexperiment in which the measurement was performed). For example, in somecases, for a cell to exhibit a phenotype of resistance, the cell onlyneeds to be positive for MHC Class I (e.g., classical MHC Class I).

Thus, in some cases, a method of predicting whether a target cell (or anindividual) is resistant to treatment with an anti-CD47/SIRPA agentincludes measuring the expression level of Major HistocompatibilityComplex (MHC) Class I (e.g., in a biological sample of the individualthat contains an inflicted cell), determining that the target cell (oran inflicted cell of the individual) is positive for MHC Class I (e.g.,classical MHC Class I), and predicting that the target cell (orindividual) is resistant to treatment with an anti-CD47/SIRPA agent. Insome cases, a method of predicting whether a target cell (or anindividual) is resistant to treatment with an anti-CD47/SIRPA agentincludes measuring the expression level of Major HistocompatibilityComplex (MHC) Class I (e.g., in a biological sample of the individualthat contains an inflicted cell), determining that the target cell (oran inflicted cell of the individual) expresses an increased level of MHCClass I (e.g., classical MHC Class I) compared to a control value, andpredicting that the target cell (or individual) is resistant totreatment with an anti-CD47/SIRPA agent.

In some cases, the level of MHC Class I expression is predictive of howresistant a cell (or an individual) is to an anti-CD47/SIRPA agent.Thus, in some cases, the method is a method of predicting the level ofresistance of a target cell (or an individual) to treatment with ananti-CD47/SIRPA agent, and the method can include: measuring theexpression level of Major Histocompatibility Complex (MHC) Class I(e.g., in a biological sample of the individual that contains aninflicted cell), comparing the measured level of MHC Class I with acontrol value, and predicting that level of resistance of the targetcell (or individual) to treatment with an anti-CD47/SIRPA agent.

In some cases, a method of predicting whether a target cell (or anindividual) is resistant to treatment with an anti-CD47/SIRPA agentincludes measuring the expression level of Major HistocompatibilityComplex (MHC) Class I (e.g., in a biological sample of the individualthat contains an inflicted cell), determining that the target cell (oran inflicted cell of the individual) is negative for MHC Class I (e.g.,classical MHC Class I), and predicting that the target cell (orindividual) is not resistant to (i.e., is susceptible to) treatment withan anti-CD47/SIRPA agent. In some cases, a method of predicting whethera target cell (or an individual) is resistant to treatment with ananti-CD47/SIRPA agent includes measuring the expression level of MajorHistocompatibility Complex (MHC) Class I (e.g., in a biological sampleof the individual that contains an inflicted cell), determining that thetarget cell (or an inflicted cell of the individual) expresses adecreased level of MHC Class I (e.g., classical MHC Class I) compared toa control value, and predicting that the target cell (or individual) isnot resistant to (i.e., is susceptible to) treatment with ananti-CD47/SIRPA agent.

In some cases, when a prediction of resistance is made, the methodfurther includes treating the individual (i.e., contacting the targetcell(s)) with an anti-MHC Class I/LILRB1 agent and an anti-CD47/SIRPAagent (e.g., co-administration to the individual, contacting the targetcell with a phagocytic cell in vitro in the presence of an anti-MHCClass I/LILRB1 agent and an anti-CD47/SIRPA agent, etc.).

The terms “determining”, “measuring”, “evaluating”, “assessing,”“assaying,” and “analyzing” are used interchangeably herein to refer toany form of measurement, and include determining if an element ispresent or not. These terms include both quantitative and/or qualitativedeterminations. Measuring may be relative or absolute. For example,“measuring” can be determining whether the expression level is less thanor “greater than or equal to” a particular threshold, (the threshold canbe pre-determined or can be determined by assaying a control sample). Onthe other hand, “measuring to determine the expression level” can meandetermining a quantitative value (using any convenient metric) thatrepresents the level of expression (i.e., expression level, e.g., theamount of protein and/or RNA, e.g., mRNA) of a particular biomarker. Thelevel of expression can be expressed in arbitrary units associated witha particular assay (e.g., fluorescence units, e.g., mean fluorescenceintensity (MFI)), or can be expressed as an absolute value with definedunits (e.g., number of mRNA transcripts, number of protein molecules,concentration of protein, etc.). Additionally, the level of expressionof a biomarker can be compared to the expression level of one or moreadditional genes (e.g., nucleic acids and/or their encoded proteins) toderive a normalized value that represents a normalized expression level.The specific metric (or units) chosen is not crucial as long as the sameunits are used (or conversion to the same units is performed) whenevaluating multiple biological samples from the same individual (e.g.,biological samples taken at different points in time from the sameindividual). This is because the units cancel when calculating afold-change in the expression level from one biological sample to thennext (e.g., biological samples taken at different points in time fromthe same individual).

The term “measuring” is used herein to include the physical steps ofmanipulating a biological sample to generate data related to the sample.As will be readily understood by one of ordinary skill in the art, abiological sample must be “obtained” prior to assaying the sample. Thus,the term “measuring” implies that the sample has been obtained. Theterms “obtained” or “obtaining” as used herein encompass the act ofreceiving an extracted or isolated biological sample. For example, atesting facility can “obtain” a biological sample in the mail (or viadelivery, etc.) prior to assaying the sample. In some such cases, thebiological sample was “extracted” or “isolated” from an individual byanother party prior to mailing (i.e., delivery, transfer, etc.), andthen “obtained” by the testing facility upon arrival of the sample.Thus, a testing facility can obtain the sample and then assay thesample, thereby producing data related to the sample. In some cases, themeasured expression level of MHC Class I is normalized (e.g., to aninternal experimental control).

The terms “obtained” or “obtaining” as used herein can also include thephysical extraction or isolation of a biological sample from a subject.Accordingly, a biological sample can be isolated from a subject (andthus “obtained”) by the same person or same entity that subsequentlyassays the sample. When a biological sample is “extracted” or “isolated”from a first party or entity and then transferred (e.g., delivered,mailed, etc.) to a second party, the sample was “obtained” by the firstparty (and also “isolated” by the first party), and then subsequently“obtained” (but not “isolated”) by the second party. Accordingly, insome embodiments, the step of obtaining does not comprise the step ofisolating a biological sample.

In some embodiments, the step of obtaining comprises the step ofisolating a biological sample (e.g., a pre-treatment biological sample,a post-treatment biological sample, etc.). Methods and protocols forisolating various biological samples (e.g., a blood sample, a serumsample, a plasma sample, a biopsy sample, an aspirate, etc.) will beknown to one of ordinary skill in the art and any convenient method maybe used to isolate a biological sample.

In subject methods, the expression level of a gene product (e.g., abiomarker) in a biological sample is measured (i.e., “determined”). By“expression level” it is meant the level of gene product (e.g. theabsolute and/or normalized value determined for the protein expressionlevel, and/or the RNA expression level of a biomarker). The term “geneproduct” or “expression product” are used herein to refer to the proteinproducts or RNA transcription products (RNA transcripts, e.g. mRNA, anunspliced RNA, a splice variant mRNA, and/or a fragmented RNA) of agene, including mRNA, and the polypeptide translation products of suchRNA transcripts. A gene product can be, for example, a protein, apost-translationally modified polypeptide, a splice variant polypeptide,an unspliced RNA, an mRNA, a splice variant mRNA, a microRNA, afragmented RNA, etc.

The biomarker used herein is MHC Class I (e.g., classical MHC Class I).Thus, “measuring the expression level” generally entails measuring theexpression level of MHC Class I (e.g., classical MHC Class I) on or in acell. In some cases, the methods include measuring the expression levelof MHC Class I (e.g., classical MHC Class I) on the surface of a cell(e.g., via flow cytometry). In some cases, the methods include measuringthe expression level of MHC Class I (e.g., classical MHC Class I) in acell (e.g., via Western Blot, ELISA assay, mass spectrometry, etc).

For measuring protein levels, the amount or level of a polypeptide inthe biological sample is determined, e.g., the protein/polypeptideencoded by the biomarker gene. In some cases, the surface protein levelis measured. In some cases, the cells are removed from the biologicalsample (e.g., via centrifugation, via adhering cells to a dish or toplastic, etc.) prior to measuring the expression level. In some cases,the intracellular protein level is measured (e.g., by lysing the cellsof the biological sample to measure the level of protein in the cellularcontents). In some cases, cells of the biological sample are identifiedas target cells (e.g., inflicted cells) (e.g., via cell sorting, viamicroscopic evaluation, via marker analysis, etc.) prior to measuringthe expression level of MHC Class I. In some cases, cells of thebiological sample are identified as target cells simultaneous withmeasuring the expression level of MHC Class I (e.g., via flowcytometry),In some cases, surface levels of MHC Class I can be measuredby extracting or otherwise enriching for or purifying surface proteins,prior to the measuring.

In some instances, the expression level of one or more additionalproteins may also be measured, and the level of biomarker expressioncompared to the level of the one or more additional proteins to providea normalized value for the biomarker expression level. Any convenientprotocol for evaluating protein levels may be employed wherein the levelof one or more proteins in the assayed sample is determined.

While a variety of different manners of assaying for protein levels areknown to one of ordinary skill in the art and any convenient method maybe used, representative methods include but are not limited toantibody-based methods (e.g., flow cytometry, ELISA, Western blotting,proteomic arrays, xMAPTM microsphere technology (e.g., Luminextechnology), immunohistochemistry, flow cytometry, and the like); aswell as non antibody-based methods (e.g., mass spectrometry).

When a prediction is made in the subject methods, the methods include astep of providing the prediction. The term “providing a prediction” isnot simply a mental step, but instead includes the active step ofreporting the prediction either by generating or report, or by orallyproviding the prediction. In some cases the prediction is provided as areport. Thus, in some instances, the subject methods may further includea step of generating or outputting a report providing the results of theevaluation of the sample, which report can be provided in the form of anon-transient electronic medium (e.g., an electronic display on acomputer monitor, stored in memory, etc.), or in the form of a tangiblemedium (e.g., a report printed on paper or other tangible medium). Anyform of report may be provided, e.g. as known in the art or as describedin greater detail below.

In some embodiments, a report is generated. A “report,” as describedherein, is an electronic or tangible document which includes reportelements that provide information of interest relating to the assessmentof a subject and its results. In some embodiments, a subject reportincludes the measured test value that represents the measured expressionlevel of MHC Class I (e.g., the normalized measured expression level).In some embodiments, a subject report includes an artisan's assessment,e.g. a prediction of resistance or susceptibility, a treatmentrecommendation, a prescription, etc. A subject report can be completelyor partially electronically generated. A subject report can furtherinclude one or more of: 1) information regarding the testing facility;2) service provider information; 3) patient data; 4) sample data; 5) anassessment report, which can include various information including: a)reference values employed, and b) test data, where test data caninclude, e.g., a protein level determination; 6) other features.

In some embodiments, a prediction is provided by generating a writtenreport. Thus, the subject methods may include a step of generating oroutputting a report, which report can be provided in the form of anelectronic medium (e.g., an electronic display on a computer monitor),or in the form of a tangible medium (e.g., a report printed on paper orother tangible medium). Any form of report may be provided.

The report may include a sample data section, which may provideinformation about the biological sample analyzed in the monitoringassessment, such as the source of biological sample obtained from thepatient (e.g. Tumor, blood, saliva, or type of tissue, etc.), how thesample was handled (e.g. storage temperature, preparatory protocols) andthe date and time collected. Report fields with this information cangenerally be populated using data entered by the user, some of which maybe provided as pre-scripted selections (e.g., using a drop-down menu).The report may include a results section. For example, the report mayinclude a section reporting the results of a marker expression leveldetermination assay, or a prediction of resistance or susceptibility.

Kits

Also provided are kits for use in the methods. The subject kits caninclude an anti-MHC ClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent.In some embodiments, a kit comprises two or more anti-MHC ClassI/LILRB1agents and/or two or more anti-CD47/SIRPA agents. In some embodiments,an anti-MHC ClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent isprovided in a dosage form (e.g., a therapeutically effective dosageform, a sub-therapeutic dosage form, e.g., in the case of ananti-CD47/SIRPA agent). In the context of a kit, an anti-MHCClassI/LILRB1 agent and/or an anti-CD47/SIRPA agent can be provided inliquid or sold form in any convenient packaging (e.g., stick pack, dosepack, etc.). The agents of a kit can be present in the same or separatecontainers. For example, a kit may have an anti-MHC ClassI/LILRB1 agentin one container and an anti-CD47/SIRPA agent in another container. Theagents may also be present in the same container.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), flash drive, and the like, on which the information has beenrecorded. Yet another form of these instructions that may be present isa website address which may be used via the internet to access theinformation at a removed site.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

EXAMPLES Example 1

The experiments here show that MHC class I, which is among the mostintensely studied regulatory complexes in immunology due to its functionin signaling to T and NK cells, plays a previously unappreciated role inprotecting cells from attack by macrophages. The results demonstratethat MHC class I expression inhibits macrophage-mediated phagocytosis ofcells, both in vitro and in vivo. Macrophage recognition of MHC class Iis shown to be specifically mediated by the surface receptor LILRB1.Biologic anti-LILRB1 agents, aimed either at MHC class I or at LILRB1,are sufficient to disrupt this protection, thus defining the MHC/LILRB1signaling axis as an important mediator of innate immune regulation anda target for therapy (e.g., anti-cancer immunotherapy).

Materials and Methods

Cell Culture

DLD-1, NCI-H69, NCI-H82, NCI-H1688, NCI-H196, NCI-H524, Bon, and KWNO1cells were grown in RPMI+GlutaMax (Life Technologies) supplemented with10% fetal bovine serum (Hyclone), and 100 U/mL penicillin andstreptomycin (Life Technologies). NCI-H128 was grown in RPMI+GlutaMax(Life Technologies) supplemented with 20% fetal bovine serum (Hyclone),and 100 U/mL penicillin and streptomycin (Life Technologies). HT-29,SkBr3, and SkMel3 were grown in McCoy's 5A+GlutaMax (Life Technologies)supplemented with 10% fetal bovine serum (Hyclone), and 100 U/mLpenicillin and streptomycin (Life Technologies). LS-174T, MCF7, andSkMel28 were grown in Eagle's Minimum Essential Media (ATCC)supplemented with GlutaMax (Life Technologies) 10% fetal bovine serum(Hyclone), and 100 U/mL penicillin and streptomycin (Life Technologies).When necessary, cells were detached from plates and disaggregated usingTrypLE Express (Life Technologies) according to the manufacturer'sindications. All cell lines were obtained from ATCC with the exceptionof KWNO1, which was a generous gift from Geoff Krampitz at StanfordUniversity. Unless otherwise indicated, cell lines were propagated andsubcultured according to ATCC guidelines.

Generation of Lentiviral Particles

HIV-based replication incompetent lentiviral particles were generated in293 Lenti-X cells (Clontech) by co-transfection of pMDG.2 vector(Addgene), psPAX2 (Addgene), and a third vector specific to thelentiviral application, using the Xtremegene HD transfection reagent(Roche) according to the manufacturer's protocol. Vectors weretransfected at a mass ratio of 4:2:1, lenti-specific vector: psPAX2:pMDG.2. After transfection, cell culture media supernatant was collectedat 36 hours and 60 hours. Lentiviral particles were concentrated eitherby ultracentrifugation for 2.5 hours at 50,000 g, or with PEG-it(Systems Biosciences) according to the manufacturer's indications.Proper biosafety and disposal techniques were followed whenever usinglentiviral reagents, according to Stanford University guidelines.

GFP-Luciferase Transduction

In order to facilitate both the FACS-based phagocytosis assay and invivo imaging, sublines of DLD1, HT-29, LS-174T, SkBr3, Bon, KWNO1,SkMel28, and SkMel3 were generated that were engineered to stablyexpress a GFP-luciferase fusion protein (Systems Biosciences, catalognumber BLIV100PA/VA-1). U2OS and SAOS2 were engineered to stably expressan RFP-luciferase fusion protein (Systems Biosciences, catalog numberBLIV101PA/VA-1). Parental, unmodified cells were harvested insingle-cell suspension and mixed with pre-warmed growth media,concentrated lentivirus, and 10 ug/ml polybrene (Sigma). Cells were thencentrifuged at 800 rpm, room temperature for 1 hour. Uniform GFP+ orRFP+ populations were then generated by sequential rounds of cellsorting on a FACSAria II cell sorter (BD Biosciences).

Macrophage Generation

Leukocyte reduction system (LRS) chambers from anonymous donors wereobtained from the Stanford Blood Center. Monocytes were purified fromthese samples on an autoMACS Pro Separator (Miltenyi) using anti-CD14microbeads optimized for whole blood separation (Miltenyi) according tothe manufacturer's suggested protocol. Monocytes were thendifferentiated to macrophages by 7-10 days of culture in IMDM+GlutaMax(Life Technologies) supplemented with 10% AB Human Serum (LifeTechnologies) and 100 U/ml penicillin and streptomycin (LifeTechnologies). NSG macrophages were generated as previously described.Briefly, bone marrow cells were harvested from the lower limbs of 6-8week old NSG mice, and cultured for 7 days in IMDM+GlutaMax¹³ (LifeTechnologies) supplemented with 10% fetal bovine serum (Hyclone), 100U/mL penicillin and streptomycin, and 10 ng/mL murine M-CSF (Peprotech).

FACS-Based Phagocytosis Assay

Each phagocytosis reaction reported in this work was performed byco-culture of 100,000 target cells and 50,000 macrophages for two hoursin ultra-low attachment 96 well U-bottom plates (Corning) inIMDM+GlutaMax (Life Technologies) without antibiotics or serum added.Macrophages were generated as described above, and harvested from platesusing TrypLE Express (Life Technologies). Target cells were eitherengineered to stably express GFP or RFP fluorescent protein, asdescribed above, or stained with Calcein AM (Life Technologies)according to the manufacturer's indications prior to co-culture.Treatment antibodies, including anti-CD47 clone Hu5F9-G4, cetuximab(Bristoll-Myers Squibb), anti-LILRB1 clone GHI/75 (BioLegend), andanti-LILRB2 clone 27D2 (Biolegend) were added to reactions at aconcentration of 10 ug/ml. After co-culture, reactions were stained werestained with APC-labeled anti-CD45 clone H130 (BioLegend) to identifyhuman macrophages, and with PE-Cy7-labeled anti-F4/80 clone BM8(BioLegend) to identify NSG mouse macrophages. DAPI staining was used toexclude dead cells from the analysis (Sigma). Reactions were run on anLSRFortessa Analyzer outfitted with a high-throughput auto-sampler (BDBiosciences). Phagocytosis was evaluated as the percentage of GFP+macrophages using FlowJo v.9.4.10 (Tree Star) and was normalized asindicated in the figure legends.

Antibody Array

The LegendScreen antibody array system (BioLegend) was used to assessthe surface phenotype of the NCI-H69, NCI-H82, NCI-H524, and NCI-H196cell lines. The cells were harvested and disaggregated with TrypLE (LifeTechnologies), and NCI-H82 and NCI-H69 were stained using Calcein AM(Life Technologies) according to the manufacturer's protocol. NCI-H82(calcein-stained) and NCI-H524 (unstained) cells were run together in amultiplexed fashion, as were NCI-H69 (calcein-stained) and NCI-H196(unstained). Cells were distributed amongst antibody-containing wells,stained, and washed according to the manufacturer's indications. Sampleswere subsequently run on an LSRFortessa Analyzer outfitted with ahigh-throughput auto-sampler (BD Biosciences). Fluorescence levels wereevaluated using FlowJo v.9.4.10 (Tree Star). Calcein staining signal wasused to deconvolute multiplexed samples.

Antibody Staining

FACS analysis was performed either on a FACSAria II cell sorter (BDBiosciences) or on an LSRFortessa Analyzer (BD Biosciences). SurfaceCD47 levels were assessed by antibody staining with clone B6H12(BioLegend) at a dilution of 1:100. HLA-A/B/C was assessed by antibodystaining with clone W6/32 (BioLegend) at a dilution of 1:50. LILRB1 wasassessed by antibody staining with clone GHI/75 (BioLegend) at adilution of 1:25. LILRB2 was assessed by antibody staining with clone27D2 (BioLegend) at a dilution of 1:25. All stains were performed on icefor 30 minutes, then washed and resuspended according to standardpractice.

DLD1 Genetic Modifications

Unmodified DLD1 cells (ATCC) were transduced with lentivirus to inducestable expression of GFP-luciferase fusion protein (Systems Biosciences,catalog number BLIV100PA/VA-1), as described above and sorted for purityusing a FACSAria II cell sorter (BD Biosciences). Sequential geneticchanges were introduced into this GFP-luciferase+ parental line.Wild-type human B2M (NC_000015.10), wild-type mouse B2m (NC_000068.7),or chimeric human-mouse B2M (hmcB2M; see below for sequence) were clonedinto the NheI and NotI sites of the pCDH-CMV-MCS-EF1-Puro vector(Systems Biosciences), and these vectors were used to producelentivirus, as described above. DLD1 cells were transduced with theseviral particles to produce DLD1-Tg(B2M), DLD1-Tg(mB2m), andDLD1-Tg(hmcB2M), respectively. DLD1-Δ(CD47) was generated by transientco-transfection of CD47-targeting TALEN vectors, described below, usingXtremegene HD (Roche) according to the manufacturer's indicatedprotocol, stained for CD47 expression using antibody clone B6H12(BioLegend), and sorted for purity using a FACSAria II cell sorter (BDBiosciences). DLD1-Tg(B2M)-Δ(CD47) was generated by transduction ofhuman B2M-encoding lentivirus into the DLD1-Δ(CD47) sub-line.

TALEN Design and Construction

TALENs were designed and assembled as described²⁹. The genomic locus ofhuman CD47 (NC_000003.12) was scanned for putative TALEN binding pairs.Exon 2 was ultimately selected for targeting and the TALEN pairsTGTCGTCATTCCATGCTTTG (SEQ ID NO: 15) and TATACTTCAGTAGTGTTTTG (SEQ IDNO: 16) were respectively cloned into the pTALEN backbone.

Human-Mouse Chimeric B2M

Chimeric B2M was designed to incorporate C-terminal amino aciddifferences from mouse B2m into a primarily human B2M sequence. Thesequence was chemically synthesized as follows (IDT), and cloned intothe NheI and NotI sites of pCDH-CMV-MCS-EF1-Puro. Sequence of mouseorigin is in lower case.

>hmcB2M_geneblock (SEQ ID NO: 17)TTTAAGCTAGCATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCtgcagagttaagcatgccagtatggccgagcccaagaccgtctactgggatc gagacatgtgaGCGGCCGCAATTT

Generation of W6/32 Fab Fragments

W6/32 antibody (BioXcell) was desalted into a solution of 20 mM sodiumcitrate pH 6.0, 25 mM cysteine, 5 mM EDTA, and diluted to aconcentration of 4 mg/mL. Protease digestion was achieved by mixing with250 ul immobilized ficin resin (Thermo Scientific) per mL of antibody.The mixture was incubated with rotation at 37° C. for 5 hours. Afterincubation, the resulting digestion reaction was passed over a monoQcolumn, and the flow-through was collected and filtered through aSuperdex-200 column. Fab fragments were quantified by Nanodrop, andchecked for purity by coomassie stain.

Crystal Structure Images

Crystal structure images were generated with MacPyMol v. 1.7.0.3 fromthe published structure IP7Q.

Mice

Nod.Cg-Prkdc^(scid) IL2rg^(tm1Wjl)/SzJ (NSG) mice were used for all invivo experiments. Mice were engrafted with tumors at approximately 6-10weeks of age, and experiments were performed with an age and sex-matchedcohort of 15 mice. Mice were maintained in a barrier facility under thecare of the Stanford Veterinary Services Center and handled according toprotocols approved by the Stanford University Administrative Panel onLaboratory Animal Care.

In Vivo Competition of DLD1 Sublines

DLD1 cells and DLD1-Tg(hmcB2M) cells were generated as detailed above.Cells were harvested from culture, counted, mixed in equal quantities,and FACS analyzed on a FACSAria II cell sorter (BD Biosciences) toconfirm approximately equal representation. Cells were then mixed with asolution of 25% low-protein matrigel (BD Biosciences) and 75%unsupplemented RPMI (Life Technologies) to a concentration of 1×10⁶cells/mL. Mice were injected subcutaneously in their right flank with100 pL of cell suspension (100,000 cells), and randomly assigned to ananalysis time-point using the list randomization tools atwww.random.org. At 7 days, 14 days, and 28 days, 5 mice per time pointwere sacrificed according to Stanford Administrative Panel on LaboratoryCare guidelines. Tumors were dissected from surrounding mouse tissuewith the aid of a Leica M165 fluorescent dissection microscope (Leica),and dissociated as previously described. Single cell suspensions werewashed, stained with W6/32 antibody as described above, and analyzedusing either a FACSAria II cell sorter or an LSRFortessa Analyzer (BDBiosciences).

Results

When donor-derived human macrophages are co-cultured with cancer cells,treatment with the humanized anti-CD47 antibody (Hu5F9-G4) induced asignificant increase in macrophage phagocytosis for a majority (12 of18) of cell lines spanning a wide variety of tumor types. However, somelines did not significantly respond to anti-CD47 therapy (6 of 18), andfurthermore, amongst those with significant responses the magnitude ofinduced phagocytosis differed widely. These differences did notcorrelate with differences in surface CD47 levels, which were high inall cell lines tested.

Based on this observation, the resistant cell lines were hypothesized toexpress one or more dominant “don't eat me” signals in addition to CD47.In order to identify these signals, an antibody array system was used tocharacterize the surface immunophenotype of four small cell lung cancerlines (NCI-H69, NCI-H524, NCI-H82, and NCI-H196) that span a broadspectrum of response to Hu5F9-G4 treatment, from highly sensitive toalmost completely resistant. The most resistant cell line of this panel,NCI-H196, expressed high levels of the surface molecules HLA-A/B/C andB2M.

HLA alpha chains and B2M protein assemble to form the classical MHCclass I complex, which has critical roles in T cell and NK cellregulation. Analysis of HLA-A/B/C expression in an extended panel of 18cell lines revealed a striking and highly significant inversecorrelation between surface levels of MHC class I and sensitivity tomacrophage-mediated phagocytosis upon Hu5F9-G4 treatment (R²=0.411,p=0.002).

In order to investigate whether MHC class I expression directly confersa functional resistance to macrophage-mediated phagocytosis, a series ofgenetic experiments were designed and executed that utilize the coloncancer line DLD1. This cell line expresses CD47 but is fully negativefor surface MHC class I expression due to biallelic genetic inactivationof the B2M locus. Surface MHC class I expression can be experimentallyrestored by lentiviral expression of wild-type B2M, whereas CD47expression can be eliminated through TALEN-induced mutation of the CD47locus. Thus, by sequential genetic modifications, sub-lines of DLD1 weregenerated with all four possible permutations of positive or negativeMHC class I and CD47 expression.

A comparison of parental DLD1 and B2M reconstituted DLD1-Tg(B2M) cellsrevealed that restoration of surface MHC class I was sufficient tosignificantly protect cells from Hu5F9-G4-induced macrophagephagocytosis (p<2×10⁻⁵). Analysis of the full allelic series of DLD1demonstrated that double-negative cells lacking both CD47 and MHCexpression were the most vulnerable to phagocytosis upon treatment withthe anti-EGFR antibody cetuximab, while expression of MHC class I wassignificantly protective (p<0.001). CD47 expression inhibited macrophageattack to an even greater degree (p<0.001). These results demonstratethat MHC class I is an important inhibitor of macrophage phagocytosis,particularly under conditions of compromised CD47 signaling.

These data suggested that in cell lines expressing high levels of MHC,disruption of this signaling axis might potentiate phagocytosis. To thisend, a fragment of antigen binding (Fab) from the pan-HLA-A/B/Cmonoclonal antibody W6/32¹⁹ was generated. Unlike the intact antibody,the W6/32 Fab binds MHC class I without introducing opsonization in theform of Fc, allowing the examination of the consequences of its blockingfunction in isolation. The anti-HLA-A/B/C Fab did not significantlyincrease phagocytosis on its own for any cell lines tested, nor did itincrease the Hu5F9-G4-induced phagocytosis of the MHC-negative cell lineDLD1. Nonetheless, in a panel of cell lines expressing high levels ofMHC co-treatment with the HLA-binding W6/32 fab significantly increasedthe effectiveness of anti-CD47 antibody for the majority of lines. Theseresults demonstrate that MHC-blocking agents can potentiatemacrophage-mediated attack of otherwise resistant cancers.

In order to further understand the mechanism by which macrophages mightdetect the MHC expression status of target cells, the identity of thereceptor or receptors involved in this process were sought. Monocytelineages have been reported to express a number of MHC-binding proteins,notably members of the LILRA and LILRB family. Structural studiessuggest that amongst this family, only two genes, LILRB1 and LILRB2,possess both MHC binding capacity and the ITIM motifs involved inintracellular transduction of repressive signaling that would benecessary to account for our experimental results.

FACS analysis of freshly isolated CD14+ human peripheral blood monocytesfrom four independent donors revealed that these cells express bothLILRB1 and LILRB2 to some extent. However, after 7 days of ex vivodifferentiation into mature macrophages, these same cell populationssignificantly increased their expression of LILRB1 but lost expressionof LILRB2 (p<0.001). This observation implicates LILRB1—but notLILRB2—as a candidate mediator of repressive MHC signaling inmacrophages.

It was therefore tested whether a LILRB1 blocking antibody could inducemacrophages to phagocytose MHC-expressing cells. Indeed, whilereconstitution of DLD1 with wild-type B2M significantly protected thesecells from Hu5F9-G4-directed macrophage attack, disrupting either LILRB1(using blocking antibody GHI/75) or HLA-A/B/C (using blocking W6/32 fab)was sufficient to facilitate phagocytosis and completely eliminate theprotective effect of MHC expression. Conversely, treatment with a LILRB2blocking antibody, 27D2, had no significant effect on Hu5F9-G4-directedphagocytosis.

All phagocytosis assays reported in this study utilized macrophagesderived from multiple independent biological donors, with no prioranalysis or selection based on HLA haplotype; despite this, no evidenceof a polymorphism-based variable response to MHC-mediated protection wasuncovered. This suggests that macrophages employ a different paradigm ofMHC detection than that utilized by T cells and NK cells. Consistentwith this observation, analysis of the crystal structure of LILRB1 boundto the human MHC class I complex revealed that the majority of aminoacid contacts between LILRB1 and MHC are within the invariant B2Msubunit, rather than the highly polymorphic HLA alpha chain.

By contrast to HLA alpha chains, human B2M is minimally polymorphicbetween individuals, but alignment of the human and mouse B2M proteinsequences reveals ˜30% mismatch, including a number of residues in thepredicted interface between B2M and LILRB1. Given this difference, itwas tested whether mouse B2M expression could endow protection againsthuman macrophage attack. Mouse B2M overexpression in human DLD1 cellsfailed to deter phagocytosis by human macrophages, but this result wasconfounded by the low efficiency with which mouse B2M returned stableMHC complexes to the cell surface. To circumvent this technicallimitation, a human-mouse chimeric B2M (hmcB2M) was generated that isprimarily human but is mutated to include eight C-terminal amino acidsof mouse sequence in the region predicted to interact with LILRB1.Expression of hmcB2M induces robust surface expression of HLA-A/B/Ccomparable to fully human B2M, but unlike human B2M it has no protectiveeffect against attack by human macrophages. This result geneticallydemonstrates that the B2M/LILRB1 interface is critical for macrophagedetection of MHC. In contrast, when DLD1 sublines were co-cultured withmouse macrophages, hmcB2M was the only tested version of B2M thatsignificantly protected from phagocytosis.

To test the in vivo consequences of MHC signaling on macrophagephagocytosis, and in particular, to determine whether MHC expression bycancer cells could confer a competitive advantage over MHC negativecancer cells in vivo, NSG (NOD-SCID II2rγ^(−/−)) mice, which producefunctional macrophages but lack functional T, B and NK cells, wereutilized. NSG mice were subcutaneously injected with a mixed populationof cells comprising 50% parental (MHC-) DLD1 and 50%hmcB2M-reconstituted (MHC+) DLD1. Although engrafted tumors initiallycontained approximately equal percentages of MHC+ andMHC− cells(pre-injection and day 7), after several weeks of growth, the tumorswere almost uniformly MHC positive (day 28). This implies thatMHC-expressing cells are subject to a significantly reduced degree ofmacrophage immunosurveillance in vivo. This is unlikely to be acell-autonomous advantage, as the hmcB2M-reconstituted DLD1 sub-linedoes not show any significant difference in growth kinetics in vitrowhen compared to parental DLD1 cells. Moreover, administration ofHu5F9-G4 (anti-CD47 antibody) slowed the growth of DLD1 cells in vivo(which are MHC− due to lack of B2M expression), but had no significanteffect on DLD1-Tg (hmcB2M) cells, which express B2M and are MHC+.

Example 2

The results presented here demonstrate that MHC class I expression bycancer cells directly inhibits macrophage-mediated phagocytosis, andfurther show that macrophage detection of MHC class I is mediated by theinhibitory surface receptor LILRB1. Biologic agents aimed either at MHCclass I or at LILRB1 were sufficient to disrupt this protection andpotentiate macrophage attack in vitro and in vivo, thus defining theMHC:LILRB1 signaling axis as not only an important regulator ofmacrophage effector function, but also a biomarker for therapeuticresponse to anti-CD47 agents, and a target for anti-cancerimmunotherapy. Some of the results presented in example 2 are alsopresented in Example 1 above.

Results

Expression of MHC Class I Correlates with Resistance to MacrophagePhagocytosis

Drugs against the CD47:SIRPA pathway have broad efficacy to inducephagocytosis of cancer cells, largely irrespective of disease subtype ortissue of origin. Accordingly, when donor-derived human macrophages wereco-cultured with a variety of solid tumor-derived cell lines, thehumanized anti-CD47 antibody Hu5F9-G4 induces a significant increase inmacrophage phagocytosis for the majority of cells (FIG. 1B, 12 of 18lines, p<0.05), as assessed by a thoroughly validated flowcytometry-based phagocytosis assay (FIG. 6) (see Liu et al, PLoS One.2015 Sep. 21; 10(9):e0137345, which is hereby incorporated by referencein its entirety, e.g., for its teachings related to anti-CD47antibodies). However, some lines do not significantly respond toanti-CD47 therapy (6 of 18), and amongst responders, the magnitude ofinduced phagocytosis varies widely (FIG. 1B). These differences did notcorrelate with cancer subtype, nor did they correlate with surface CD47levels, which were high in all cell lines tested (FIG. 7). BecauseHu5F9-G4 simultaneously blocks CD47:SIRPA signaling and opsonizes cells,we speculated that Hu5F9-G4-resistant lines must express one or more“don't eat me” signals in addition to CD47.

In order to identify these signals, an antibody array system was used tocharacterize the surface immunophenotype of five cell lines: the coloncancer lines DLD1 and HCT116; the small cell lung cancer lines NCI-H82and NCI-H196; and the pancreatic neuroendocrine tumor line KWNO1. Theselines span a broad spectrum of tumor types, as well as a wide range ofsensitivity to phagocytosis upon Hu5F9-G4 treatment (FIG. 1B). Inanalyzing the data from these arrays, an intriguing relationship betweenexpression of MHC class I proteins and resistance to Hu5F9-G4-inducedmacrophage phagocytosis was noted (FIG. 8).

HLA alpha chains and the B2M protein assemble to form the MHC class Icomplex, which has essential roles in T cell and NK cell regulation, butno classically described function in the regulation of macrophages.Nonetheless, analysis of HLA expression across the panel of 18 celllines revealed a highly significant correlation between surface levelsof MHC class I and resistance to macrophage-mediated phagocytosis uponHu5F9-G4 treatment (FIG. 1C, R²=0.411, p=0.002).

MHC Class I Directly Protects Cells from Macrophage Attack

In order to investigate this correlation, a series of geneticexperiments were performed utilizing the KWNO1 and DLD1 lines. Bothlines were positive for expression of CD47 (FIG. 2A, FIG. 2B), but whileKWNO1 expressed high levels of MHC class I, DLD1 was negative forsurface MHC due to biallelic inactivation of the B2M locus. Throughirreversible genetic modification and sequential rounds offluorescence-activated cell sorting (FACS), polyclonal sub-lines ofKWNO1 and DLD1 were generated with all four permutations of positive ornegative expression of MHC and CD47 (FIG. 2A and FIG. 2B).

Co-culture of these lines with donor-derived human macrophages revealedthat deletion of surface MHC was sufficient to modestly butsignificantly increase spontaneous phagocytosis in some cases (FIG. 2C,p<0.01). Furthermore, in a critical confirmation of the hypothesis, MHC−cells were significantly more sensitive to anti-CD47-inducedphagocytosis than their MHC+ counterparts (FIG. 2C, d p<0.001). Analysisof the extended allelic panels of these lines confirmed that upontreatment with opsonizing antibody, cells lacking both CD47 and MHCexpression were significantly more sensitive to phagocytosis than cellsexpressing MHC class I alone (FIG. 2E and FIG. 2F, blue versus green;p<0.05 and p<0.001, respectively) or cells expressing CD47 alone (FIG.2E and FIG. 2F, blue versus black, p<0.001), and in the KWNO1 cell line,simultaneous expression of both MHC and CD47 was significantly moreprotective than either signal alone (FIG. 2E, red, p<0.001). Thus, MHCclass I and CD47 are independent anti-phagocytic signals that can workcooperatively to protect cells from macrophage attack.

Based on this result, it was speculated that blockade of MHC signalingcould be a general tool to sensitize cells to phagocytosis. A Fabfragment derived from the pan-HLA-A, B, C monoclonal antibody, W6/32,was therefore generated, allowing the examination of the MHC blockingeffects of this antibody in isolation from any Fc-mediated effects.HLA-binding Fab did not influence the phagocytosis of a MHC-negativecell line, DLD1 (FIG. 2G, left panel). However, when applied to a panelof MHC high cells (FIG. 9), the HLA-binding Fab significantly increasedanti-CD47-induced phagocytosis for 4 out of 6 lines tested (FIG. 2G),including KWNO1, thus independently confirming the results of thegenetic experiments (FIG. 2C and FIG. 2D).

LILRB1 is the Primary Receptor for Inhibitory MHC Signaling toMacrophages

In order to further investigate the phenomenon of MHC-to-macrophagesignaling, the next goal was to identify the receptor or receptorsinvolved in its detection. FACS analysis of freshly isolated CD14+ humanperipheral blood monocytes from four independent donors revealed thatthe majority of these cells express both LILRB1 and LILRB2 (FIG. 3A,FIG. 3B). However, after 7 days of ex vivo differentiation into maturemacrophages, LILRB1 expression significantly increased (FIG. 3A, bp<0.001), while the percentage of cells expressing LILRB2 significantlydiminished (FIG. 3A, b, p<0.001). This observation strongly implicatedLILRB1—but largely excluded LILRB2—as a mediator of repressive MHCsignaling in mature human macrophages.

In order to functionally confirm this hypothesis, it was next testedwhether blocking LILRB1 could increase phagocytosis of MHC+ cells.Indeed, GHI/75, a blocking monoclonal antibody against LILRB1,significantly increased the Hu5F9-G4-induced phagocytosis specificallyof MHC+ cells (FIG. 3C, p<0.01), eliminating the differential betweenMHC+ and MHC− sub-lines for both KWNO1 (FIG. 3C) and DLD1 (FIG. 10).Conversely, treatment with a LILRB2 blocking antibody had no significanteffect on phagocytosis for any cell population (FIG. 3B and FIG. 10).Taken together, these results strongly support LILRB1 as the primaryeffector of MHC detection during regulation of phagocytosis

B2M Protein Confers Species-Specific MHC Detection by Macrophages

All assays reported in this study utilized macrophages derived frommultiple independent biological donors, with no prior analysis orselection based on HLA haplotype; despite this, a highly consistentprotection by MHC expression was observed, suggesting a differentparadigm of detection than the allele-specific mechanisms employed by Tcells and NK cells. Consistent with this result, analysis of thepreviously solved crystal structure of LILRB1 bound to the human MHCclass I complex revealed that the majority of contact residues betweenLILRB1 and MHC are within the invariant B2M subunit rather than thehighly polymorphic HLA alpha chain (FIG. 4A).

There are substantial differences in LILRB-family genes between humansand mice, but previous work suggests that the mouse receptor PirB maysubsume most of the functions described for both human LILRB1 andLILRB2, including repressive signaling upon MHC binding. Accordingly,PirB was found to be highly expressed on the surface of invitro-differentiated mouse macrophages (FIG. 11). However, in contrastto human macrophages, mouse macrophages did not strongly discriminatebetween human MHC+ andMHC− cells.

Alignment of the human and mouse B2M protein sequences revealed anidentity mismatch of approximately 30%, including several amino acids inthe region of predicted contact between human B2M and LILRB1 (FIG. 4A).Differences in these residues might account for the lack of signalingfrom human MHC to mouse macrophages, and it was next sought to determinewhether mouse B2m and human B2M could endow cells with aspecies-specific protection against phagocytosis.

An attempt to express mouse B2m in human cells revealed that thisforeign protein was unable to form stable surface MHC complexes withhuman HLA alpha chains (FIG. 12). To circumvent this technicallimitation, a human-mouse chimeric B2M (hmcB2M) was generated in whichthe human protein was mutated to include amino acids of mouse sequencewithin the predicted region of interaction with LILRB1 (FIG. 4A, insetregion). Expression of hmcB2M in either B2M-negative parental DLD1cells, or in B2M-deleted KWNO1, enabled robust surface expression of HLAcomparable to that achieved with fully human B2M protein (FIG. 4B,purple versus red).

Using these lines, it was confirmed that although human B2M stronglyprotected target cells against human macrophages (FIG. 4C, y axis, blackversus red), it was inefficient in protecting against murine macrophages(FIG. 4C, x axis, black versus red); in contrast, expression of hmcB2Mconferred precisely the inverse protection (FIG. 4C, black versuspurple). This result genetically demonstrates that the amino acidresidues in the LILRB-interacting region of B2M are critical forspecies-specific detection of MHC by both human and mouse macrophages

MHC Protects Cancer Cells from Macrophage Attack In Vivo

The next goal was to study the in vivo consequences of MHC-to-macrophagesignaling, and in particular to determine whether MHC expression bycancer cells protects them from macrophage immune surveillance in vivo.Based on the species-specific nature of this interaction (FIG. 4C), itwas a goal first to establish a xenograft system by which we couldassess the interaction of human macrophages with human cancer cells invivo. NSG (NOD-SCID II2rγ^(−/−)) mice produce functional cells of themyeloid lineage, but lack T, B and NK cells, and therefore can acceptcross-species transplants. These mice were used as hosts into which exvivo human macrophages were co-engrafted alongside KWNO1 human cancercells (schematic in FIG. 5A).

MHC+ KWNO1 cells readily engrafted into NSG hosts, despite the presenceof human macrophages (FIG. 5B, left panel), and consistent with the invitro assays, were only modestly affected by treatment with the blockinganti-CD47 antibody Hu5F9-G4 (FIG. 5B, left panel). In contrast,treatment of the MHC− KWNO1 sub-line with Hu5F9-G4 resulted in completeclearance of tumor cells, with no bioluminescence detectable at 14 dayspost-injection (FIG. 5B, right panel), a striking demonstration of thepotency of MHC-mediated macrophage regulation. In a symmetricdemonstration of this principle, combination treatment with anti-CD47and anti-LILRB1 blocking antibody was sufficient to dramatically reducetumor formation by MHC+ KWNO1 cells (FIG. 5B, left panel), leadingto >80-fold reduction in overall tumor burden by day 7 as compared toPBS treated mice, and a ˜50-fold reduction as compared to tumors treatedwith Hu5F9-G4 alone (FIG. 5B and FIG. 13).

Phagocytosis in this xenograft system had tapered by day 7, consistentwith the dispersal of the co-engrafted human macrophages or of thehuman-specific antibody treatments after initial injection (FIG. 13).Therefore, in order to study the long-term effects oftumor-to-macrophage MHC signaling in vivo, sub-lines of KWNO1 expressingthe human-mouse chimeric B2M were used (FIG. 4). An initial histologicalanalysis of tumors derived from a mixed population of MHC− cells andchimeric MHC+ cells revealed substantial infiltration by mousemacrophages (FIG. 5C, blue), as well as clear instances of tumor cellphagocytosis (FIG. 5C, white arrowhead), thereby demonstrating that NSGmacrophages are indeed present in the tumor and capable of mounting ananti-tumor response against these cells in vivo.

In order to quantitatively compare the in vivo sensitivity of MHC−versus chimeric MHC+ cells to anti-CD47 agents, MHC− KWNO1 cells orchimeric MHC+ KWNO1 cells were grafted into the flanks of NSG mice, andbioluminescent imaging was used to follow tumor growth under conditionsof PBS treatment or once-weekly Hu5F9-G4 administration (schematic inFIG. 5D). At early time points, there was no significant difference inluminescence between the tumors of any treatment group, or betweenchimeric MHC+ and MHC− tumors (FIG. 5E). By day 42, once-weeklyanti-CD47 treatment had significantly slowed the growth of both MHC− andchimeric MHC+ tumors (FIG. 5E, p<1e-4 and p<0.05, respectively).However, there was significantly less therapeutic benefit to anti-CD47for MHC+ KWNO1 tumors, which grew at a significantly faster rate thanMHC− tumors (FIG. 5E, p<0.05; see FIG. 16 for a comprehensivestatistical comparison between all time points and groups). Given thatthese cancer sub-lines differ only in their B2M expression status, whichhad no measurable effect on their in vitro growth rate (FIG. 14 and FIG.15), these results demonstrate a MHC-mediated effect on macrophageimmune surveillance of tumor cells in vivo

Discussion

The data presented here demonstrate that MHC class I is a key regulatorysignal for the effector functions of macrophages, thus expanding ourunderstanding of one of immunology's best-studied and most importantsignaling complexes, and highlighting the central role of MHC incoordinating the activity of both the adaptive and innate branches ofthe immune system. MHC:LILRB1 signaling has been previously studied in asubset of NK cells, and in the myeloid lineage for its role in monocyteactivation. However, as described here, while freshly isolated monocyteshave detectable expression of LILRB1, surface levels of this geneincrease approximately 10-fold during differentiation into maturemacrophages (FIG. 3B). This increase, when taken together with thefunctional data described here, suggests that regulation of macrophagephagocytosis is a key role of LILRB1 signaling.

The results herein have important implications for several aspects ofmacrophage biology. Viruses can avoid presentation of foreign peptidesto T cells by down-regulation of surface MHC class I, which canconsequently activate NK cell attack due to lack of MHC binding bykiller cell immunoglobulin-like receptors (KIRs). It is likely that theMHC:LILRB1 signaling axis serves an analogous role formacrophage-mediated immune surveillance of infected cells. Furthermore,the human cytomegalovirus (CMV) family has evolved to encode a protein,UL-18, which mimics HLA but prevents binding by T cells. It binds LILRB1with >1000-fold higher affinity as compared to native MHC complexes, andaccording to previous reports, inhibits the subset of NK cells thatexpress LILRB1. Given the high LILRB1 expression in mature macrophages,UL-18 is likely to modulate macrophage-mediated phagocytosis ofCMV-infected cells, and this may therefore influence the pathology ofCMV infection.

MHC:LILRB1 signaling is also important in the dynamics of programmedcell removal. As erythrocytes age, their effective CD47 signalinggradually decreases, eventually dropping below a critical threshold andthus enabling phagocytosis by macrophages. While the majority of normalhuman tissues express MHC class I, including erythrocyte precursors,mature erythrocytes lack surface MHC. In light of the results herein,this lack of expression may be a factor in priming erythrocytes foreventual phagocytosis, and may serve to in part explain the anemiainduced by anti-CD47 antibodies or CD47-binding Fc-fusion proteins whendeployed as cancer therapies.

As demonstrated by the results herein, both in vitro and in vivo, lackof MHC expression endows cancer cells with sensitivity to phagocytosis.Many cancers present clinically with compromised or negative expressionof surface MHC. Due to impaired presentation of mutated cancerneo-antigens, these patients are very poor candidates for T cell focusedtherapies, but the results presented here suggest that they are idealcandidates for macrophage-mediated immunotherapies. Thus, MHC expressionstatus may be used as predictive biomarker not only for the efficacy ofT cell therapies such as anti-PD-1 or PD-L1 agents, but also for theefficacy of anti-CD47 or anti-SIRPA drugs in a variety of diseaseindications.

Beyond its use a biomarker, the work here suggests that in patients withtumors expressing normal or high levels of MHC, therapeutic agents thatdisrupt the MHC:LILRB1 interaction may independently increase theefficacy of tumor-binding monoclonal antibodies, and will cooperate withagents against CD47:SIRPA.

Agents against the MHC:LILRB1 signaling axis can be an importantcomponent of any therapeutic regimen that aims to engage macrophages inthe fight against cancer

Experimental Procedures

Cell Culture

DLD-1, NCI-H69, NCI-H82, NCI-H1688, NCI-H196, NCI-H524, Bon, and KWNO1cells were grown in RPMI+GlutaMax (Life Technologies) supplemented with10% fetal bovine serum (Hyclone), and 100 U/mL penicillin andstreptomycin (Life Technologies). NCI-H128 was grown in RPMI+GlutaMax(Life Technologies) supplemented with 20% fetal bovine serum (Hyclone),and 100 U/mL penicillin and streptomycin (Life Technologies). HT-29,SkBr3, and SkMel3 were grown in McCoy's 5A+GlutaMax (Life Technologies)supplemented with 10% fetal bovine serum (Hyclone), and 100 U/mLpenicillin and streptomycin (Life Technologies). LS-174T, MCF7, andSkMel28 were grown in Eagle's Minimum Essential Media (ATCC)supplemented with GlutaMax (Life Technologies), 10% fetal bovine serum(Hyclone), and 100 U/mL penicillin and streptomycin (Life Technologies).When necessary, cells were detached from plates and disaggregated usingTrypLE Express (Life Technologies) according to the manufacturer'sindications. All cell lines were obtained from ATCC with the exceptionof KWNO1, which was a generous gift from Geoff Krampitz at StanfordUniversity. Unless otherwise indicated, cell lines were propagated andsubcultured according to ATCC guidelines.

Generation of Lentiviral Particles

HIV-based replication incompetent lentiviral particles were generated in293 Lenti-X cells (Clontech) by co-transfection of pMDG.2 vector(Addgene), psPAX2 (Addgene), and a third vector specific to thelentiviral application, using the Xtremegene HD transfection reagent(Roche) according to the manufacturer's protocol. Vectors weretransfected at a mass ratio of 4:2:1, lenti-specificvector:psPAX2:pMDG.2. After transfection, cell culture media supernatantwas collected at 36 hours and 60 hours. Lentiviral particles wereconcentrated either by ultracentrifugation for 2.5 hours at 50,000 g, orwith PEG-it (Systems Biosciences) according to the manufacturer'sindications. Proper biosafety and disposal techniques were followedwhenever using lentiviral reagents, according to Stanford Universityguidelines.

Generation of DLD1 and KWNO1 Sub-Lines

In order to generate sub-lines of DLD1 and KWNO1, parental, unmodifiedcells were harvested in single-cell suspension and mixed with pre-warmedgrowth media, concentrated lentivirus, and 10 μg/mL polybrene (Sigma).Cells were then centrifuged at 1800 rpm, room temperature for 45minutes. Lentiviral pools included at least three distinct viralspecies: one encoding for the Cas9 nuclease, and two others encoding fordifferent CRISPR small guide RNAs (sgRNAs) targeting the first exon ofeither CD47 or B2M, as appropriate. CRISPR sgRNAs were designed usingthe tools at genome-engineering.org, and were of the followingsequences: sgCD47-1: GCTACTGAAGTATACGTAAAG (SEQ ID NO: 18), sgCD47-2:GCTTGTTTAGAGCTCCATCAA (SEQ ID NO: 19), sgB2M-1: GAGTAGCGCGAGCACAGCTA(SEQ ID NO: 20), sgB2M-2: GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 21). 7 dayspost infection, cells were assessed for expression status by flowcytometry. CD47 was assessed by staining with APC-conjugated B6H12(Biolegend) and HLA-A, B, C was assessed by staining withPE-Cy7-conjugated W6/32 (Biolegend). Cells were sorted on a FACSAria IIcell sorter (BD Biosciences). Typically, each cell line was sorted threetimes, separated by several days of recovery and expansion betweenrounds. Wild-type human B2M (NC_000015.10), wild-type mouse B2m(NC_000068.7), or chimeric human-mouse B2M (hmcB2M; see below forsequence) were cloned into the NheI and NotI sites of thepCDH-CMV-MCS-EF1-Puro vector (Systems Biosciences), and these vectorswere used to produce lentivirus and introduce transgenes, asappropriate. DLD1-Δ(CD47) was generated by transient co-transfection ofCD47-targeting TALEN vectors, described below, using Xtremegene HD(Roche) according to the manufacturer's indicated protocol. All othergenetic modifications were induced using lentiviral delivery oftransgenes, or lentiviral delivery of Cas9 and corresponding sgRNAs, asdescribed above.

TALEN Design and Construction

TALENs were designed and assembled as described. The genomic locus ofhuman

CD47 (NC_000003.12) was scanned for putative TALEN binding pairs. Exon 2was ultimately selected for targeting and the TALEN pairsTGTCGTCATTCCATGCTTTG (SEQ ID NO: 22) and TATACTTCAGTAGTGTTTTG (SEQ IDNO: 23) were respectively cloned into the pTALEN backbone.

GFP-Luciferase Transduction

In order to facilitate both the FACS-based phagocytosis assay and invivo imaging, we generated sublines of DLD1, HT-29, LS-174T, SkBr3, Bon,KWNO1, SkMel28, and SkMel3 engineered to stably express a GFP-luciferasefusion protein (Systems Biosciences, catalog number BLIV100PA/VA-1).U2OS and SAOS2 were engineered to stably express an RFP-luciferasefusion protein (Systems Biosciences, catalog number BLIV101PA/VA-1).Parental, unmodified cells were harvested in single-cell suspension andmixed with pre-warmed growth media, concentrated lentivirus, and 10μg/mL polybrene (Sigma). Cells were then centrifuged at 1800 rpm, roomtemperature for 45 minutes. Uniform GFP+ or RFP+ populations were thengenerated by sequential rounds of cell sorting on a FACSAria II cellsorter (BD Biosciences).

Macrophage Generation

Leukocyte reduction system (LRS) chambers from anonymous donors wereobtained from the Stanford Blood Center. Monocytes were purified fromthese samples on an autoMACS Pro Separator (Miltenyi) using anti-CD14microbeads optimized for whole blood separation (Miltenyi) according tothe manufacturer's suggested protocol. Monocytes were thendifferentiated to macrophages by 7-10 days of culture in IMDM+GlutaMax(Life Technologies) supplemented with 10% AB Human Serum (LifeTechnologies) and 100 U/mL penicillin and streptomycin (LifeTechnologies). NSG macrophages were generated as previously described.Briefly, bone marrow cells were harvested from the lower limbs of 6-8week old NSG mice, and cultured for 7 days in IMDM+GlutaMax¹³ (LifeTechnologies) supplemented with 10% fetal bovine serum (Hyclone), 100U/mL penicillin and streptomycin, and 10 ng/mL murine M-CSF (Peprotech).

FACS-Based Phagocytosis Assay

Each phagocytosis reaction reported in this work was performed byco-culture of 100,000 target cells and 50,000 macrophages for two hoursin ultra-low attachment 96 well U-bottom plates (Corning) inIMDM+GlutaMax (Life Technologies) without antibiotics or serum added.Macrophages were generated as described above, and harvested from platesusing TrypLE Express (Life Technologies). Target cells were eitherengineered to stably express GFP or RFP fluorescent protein, asdescribed above, or stained with Calcein AM (Life Technologies)according to the manufacturer's indications prior to co-culture.Treatment antibodies, including anti-CD47 clone Hu5F9-G4, cetuximab(Bristoll-Myers Squibb), anti-LILRB1 clone GHI/75 (BioLegend), andanti-LILRB2 clone 27D2 (Biolegend) were added to reactions at aconcentration of 10pg/mL. After co-culture, reactions were stained werestained with APC-labeled anti-CD45 clone H130 (BioLegend) to identifyhuman macrophages, and with PE-Cy7-labeled anti-F4/80 clone BM8(BioLegend) to identify NSG mouse macrophages. DAPI staining was used toexclude dead cells from the analysis (Sigma). Reactions were run on anLSRFortessa Analyzer outfitted with a high-throughput auto-sampler (BDBiosciences). Phagocytosis was evaluated as a sum of the GFP+macrophages (both “Mid” and “High” gates were included in this number,as described in FIG. 6), expressed as a percentage of the totalmacrophages, as analyzed using FlowJo v.9.4.10 (Tree Star) and wasnormalized as indicated in the figure legends. Unless otherwise stated,each replicate represents a true biological replicate (i.e. anindependent human macrophage donor), and unless otherwise indicated,replicates were split between a minimum of two independent experimentalinstances (e.g. four independent donors evaluated on day 1, with anadditional four independent donors evaluated on day 2). Sample size waschosen to ensure a greater than 95% probability of identifying, bytwo-tailed t-test, an effect of >20%, assuming a technical variation of15%.

Antibody Array

We used the LegendScreen antibody array system (BioLegend) to assess thesurface phenotype of the NCI-H69, NCI-H82, NCI-H524, and NCI-H196 celllines. The cells were harvested and disaggregated with TrypLE (LifeTechnologies), and NCI-H82 and NCI-H69 were stained using Calcein AM(Life Technologies) according to the manufacturer's protocol. NCI-H82(calcein-stained) and NCI-H524 (unstained) cells were run together in amultiplexed fashion, as were NCI-H69 (calcein-stained) and NCI-H196(unstained). Cells were distributed amongst antibody-containing wells,stained, and washed according to the manufacturer's indications. Sampleswere subsequently run on an LSRFortessa Analyzer outfitted with ahigh-throughput auto-sampler (BD Biosciences). Fluorescence levels wereevaluated using FlowJo v.9.4.10 (Tree Star). Calcein staining signal wasused to deconvolute multiplexed samples.

Antibody Staining

FACS analysis was performed either on a FACSAria II cell sorter (BDBiosciences) or on an LSRFortessa Analyzer (BD Biosciences). SurfaceCD47 levels were assessed by antibody staining with clone B6H12(BioLegend) at a dilution of 1:100. HLA-A/B/C was assessed by antibodystaining with clone W6/32 (BioLegend) at a dilution of 1:50. LILRB1 wasassessed by antibody staining with clone GHI/75 (BioLegend) at adilution of 1:25. LILRB2 was assessed by antibody staining with clone27D2 (BioLegend) at a dilution of 1:25. All stains were performed on icefor 30 minutes, then washed and resuspended according to standardpractice.

Human-Mouse Chimeric B2M

Chimeric B2M was designed to incorporate C-terminal amino aciddifferences from mouse B2m into a primarily human B2M sequence. Thesequence (indicated below) was chemically synthesized (IDT), and clonedinto the NheI and NotI sites of pCDH-CMV-MCS-EF1-Puro. Sequence of mouseorigin is in lower case.

>hmcB2M_geneblock (SEQ ID NO: 24)TTTAAGCTAGCATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCtgcagagttaagcatgccagtatggccgagcccaagaccgtctactgggatc gagacatgtgaGCGGCCGCAATTT.Generation of W6/32 Fab Fragments

W6/32 antibody (BioXcell) was desalted into a solution of 20 mM sodiumcitrate pH 6.0, 25 mM cysteine, 5 mM EDTA, and diluted to aconcentration of 4 mg/mL. Protease digestion was achieved by mixing with250 μL immobilized ficin resin (Thermo Scientific) per mL of antibody.The mixture was incubated with rotation at 37° C. for 5 hours. Afterincubation, Fab fragments were purified from undigested antibody and Fcfragments by ion-exchange chromatography with a monoQ column, followedby size exclusion chromatography with a Superdex-200 column. Fabfragments were quantified by Nanodrop, and checked for purity bycoomassie stain.

Crystal Structure Images

Crystal structure images were generated with MacPyMol v. 1.7.0.3 fromthe published structure IP7Q.

Mice

Nod.Cg-Prkdc^(scid) IL2rg^(tm1Wjl)/SzJ (NSG) mice were used for all invivo experiments. Mice were engrafted with tumors at approximately 6-10weeks of age, and experiments were performed with age and sex-matchedcohorts. Mice were maintained in a barrier facility under the care ofthe Stanford Veterinary Services Center and handled according toprotocols approved by the Stanford University Administrative Panel onLaboratory Animal Care.

Humanized NSG Mouse Model

Ex vivo cultured human macrophages and GFP-luciferase labeled KWNO1 orKWNO1-Δ(B2M) cells and were generated and harvested as described above.200,000 human macrophages, 100,000 target cells, and 1 μL of PBS, 1 pLof Hu5F9-G4 1 mg/mL stock, or 1 μL GHI/75 1 mg/ml stock was resuspendedin 50 μL of RPMI and injected subcutaneously into the right flank of anNSG mouse. 5 mice were used for each treatment group. Tumorbioluminescence was assessed using an IVIS Spectrum imager (PerkinElmer) at 12 hours post-injection, 4 days post-injection, 7 dayspost-injection and 14 days post-injection, as indicated. Experimentswere performed in a non-blinded fashion.

In Vivo Growth Experiments

NSG mice were injected subcutaneously in the right flank with either100,000 GFP-lucif erase-labeled KWNO1-Δ(B2M) (30 mice) orKWNO1-Tg(hmcB2M)-Δ(B2M) cells (30 mice) and randomized into treatmentcohorts using the list randomization tools at random.org. Sample sizewas chosen to ensure a greater than 95% probability of identifying, bytwo-tailed t-test, an effect of >50%, assuming a technical variation of50%. Starting on day 14, mice were treated once per week byintraperitoneal injection of either 100 μL of PBS or 250 μg Hu5F9-G4 ata concentration of 2.5 mg/mL. Tumor luminescence was measured once perweek using an IVIS Spectrum imager (Perkin Elmer). Measurements werediscontinued when tumors in a measurement group began to exceed 5×10¹⁰total flux, which is, in our experience, a threshold above whichbioluminescent signals are less accurate. Across additional experiments,including pilot experiments, additional mice were engraftedsubcutaneously with these cell lines and treated with PBS, but were notincluded as part of this data set. One additional cage of 4 mice wasengrafted with each KWNO1-Δ(B2M) and KWNO1-Tg(hmcB2M)-Δ(B2M) andreceived once-weekly Hu5F9-G4 treatment, but were ultimately notincluded in the final analysis of the experiment. Mouse experiments wereperformed in a non-blinded fashion.

Histology

KWNO1 tumors were fixed in 2% paraformaldehyde overnight at fourdegrees. Tissue was embedded and frozen in optimal cutting temperaturecompound O.C.T (Sakura) or embedded in paraffin. Frozen sections werecut at 4-7 μm and saved for immunofluorescence.

Immunofluorescence

Immunofluorescence studies were performed on frozen sections. Frozensections were thawed at room temperature for ten minutes and washed inPBS twice. Slides were blocked in 5% serum for 30 minutes at roomtemperature. Sections were subsequently stained with primary antibodiesagainst F4:80 (1:100, rat monoclonal, Abcam) overnight at 4C, and washedthree times in PBS. Slides were stained were incubated with secondaryantibodies conjugated to AlexaFluor 647 for one to two hours at roomtemperature. Stains were washed once with PBST and three times with PBSbefore nuclear staining with Hoechst 33342 (Life Technologies), for twominutes and mounted with Fluoromount G (Southern Biotech). Basic photoprocessing, including fluorescence channel false-coloring, channelmerge, and brightness and contrast adjustment, were performed usingAdobe Photoshop (Adobe).

Example 3

FIG. 17. Results from experiments that measure phagocytosis of twodifferent human breast cancer cell lines (MDA-MB-468 and MDA-MB-231) byhuman macrophages with and without anti-LILRB1 antibody (and in thepresence or absence of other antibodies, e.g., Hu5F9-G4 which is ananti-CD47 antibody). Bars from left to right are: IgG₄, Hu5F9-G4,Cetuximab, Trastuzumab, Panitumumab, Cetuximab+Hu5F9-G4,Trastuzumab+Hu5F9-G4, and Panitumumab+Hu5F9-G4.

FIG. 18 presents data showing that CD47 and MHC class I signaling axesare independent anti-phagocytic signals. FACS-based measurement ofphagocytosis by human macrophages co-cultured with parental KWNO1 cells(gray) and B2M-deleted KWNO1 cells (pink), upon treatment with PBS, theanti-CD47 antibody Hu5F9-G4 (which blocks the CD47/SIRPA interaction),or the anti-CD47 antibody 2D3 (which binds CD47 but does not block itsinteraction with SIRPA, and thus represents any antibody that binds to atarget cell and opsonizes that cell). Values are normalized to the maxphagocytosis observed for a given set of replications. Deletion of B2Mand consequent elimination of surface expression of MHC class Isignificantly increases the phagocytosis of KWNO1 cells relative toparental KWNO1 cells upon opsonization with the non-blocking anti-CD47antibody 2D3 (***p<1×10⁻⁴, ANOVA), thus demonstrating that disruption ofMHC classI/LILRB1 increases phagocytosis even under conditions of intactCD47/SIRPA signaling. Co-disruption of MHC class I/LILRB1 signaling andCD47/SIRPA signaling by treatment of B2M-deleted KWNO1 cells withHu5F9-G4, which blocks CD47/SIRPA and opsonizes target cells, leads to asignificant and synergistic increase in phagocytosis relative todisruption of either signaling pathway alone (***p<1×10⁻⁴, ANOVA).

The data presented here demonstrate that MHC class I is a criticalregulatory signal for the effector functions of macrophages. Thesefindings identify the MHC/LILRB1 signaling axis as a target fortreatment (e.g., cancer immunotherapy). By disrupting this pathway,e.g., in conjunction with tumor-specific monoclonal antibodies andagents (e.g., against the CD47/LILRB1 axis), therapeutic agents againstMHC/LILRB1 may enable stimulation of potent macrophage-mediatedanti-cancer (and/or anti-chronic infection) responses.

What is claimed is:
 1. A method of inducing phagocytosis of a targethematologic cancer cell, the method comprising: contacting a targethematologic cancer cell with a macrophage in the presence of an antibodythat specifically binds to leukocyte immunoglobulin-like receptorsubfamily B member 1 (LILRB1) and does not activate signaling throughLILRB1 upon binding, and an antibody that binds to the targethematologic cancer cell and thereby opsonizes the target cell, for aperiod of time sufficient to induce phagocytosis of the target cell bythe macrophage.
 2. The method of claim 1, wherein the hematologic canceris a leukemia.
 3. The method of claim 2, wherein the leukemia is acutemyeloid leukemia (AML).
 4. The method of claim 2, wherein the leukemiais acute lymphoblastic leukemia (ALL).
 5. The method of claim 2, whereinthe leukemia is chronic myeloid leukemia (CML).
 6. The method of claim2, wherein the leukemia is chronic lymphocytic leukemia (CLL).
 7. Themethod of claim 1, wherein the hematologic cancer is a lymphoma.
 8. Themethod of claim 7, wherein the lymphoma is Hodgkin lymphoma.
 9. Themethod of claim 7, wherein the lymphoma is non-Hodgkin lymphomas (NHL).10. The method of claim 9, wherein the lymphoma is diffuse large B-Celllymphoma.
 11. The method of claim 9, wherein the lymphoma is follicularlymphoma.
 12. The method according to claim 1, wherein the contacting isin vitro or ex vivo.
 13. The method according to claim 1, wherein thecontacting is in vivo.
 14. The method according to claim 7, wherein saidcontacting is in the presence of an antibody that specifically binds toCD47 and blocks interaction of CD47 and SIRPa.
 15. The method accordingto claim 1, wherein the antibody that binds to the target cell binds toCD20.
 16. The method according to claim 15, wherein the antibody isrituximab, tositumomab or ibritumomab.
 17. The method according to claim1, wherein the antibody that binds to the target cell binds to CD52. 18.The method of claim 16, wherein the antibody is alemtuzumab.
 19. Themethod according to claim 1, wherein the antibody that binds to thetarget cell binds to CD22.
 20. The method according to claim 1, whereinthe antibody that binds to the target cell is gemtuzumab.